Assessment of the Impact of Carvedilol Administered Together with Dexrazoxan and Doxorubicin on Liver Structure and Function, Iron Metabolism, and Myocardial Redox System in Rats.

Late cardiotoxicity is a formidable challenge in anthracycline-based anticancer treatments. Previous research hypothesized that co-administration of carvedilol (CVD) and dexrazoxane (DEX) might provide superior protection against doxorubicin (DOX)-induced cardiotoxicity compared to DEX alone. However, the anticipated benefits were not substantiated by the findings. This study focuses on investigating the impact of CVD on myocardial redox system parameters in rats treated with DOX + DEX, examining its influence on overall toxicity and iron metabolism. Additionally, considering the previously observed DOX-induced ascites, a seldom-discussed condition, the study explores the potential involvement of the liver in ascites development. Compounds were administered weekly for ten weeks, with a specific emphasis on comparing parameter changes between DOX + DEX + CVD and DOX + DEX groups. Evaluation included alterations in body weight, feed and water consumption, and analysis of NADPH2, NADP+, NADPH2/NADP+, lipid peroxidation, oxidized DNA, and mRNA for superoxide dismutase 2 and catalase expressions in cardiac muscle. The iron management panel included markers for iron, transferrin, and ferritin. Liver abnormalities were assessed through histological examinations, aspartate transaminase, alanine transaminase, and serum albumin level measurements. During weeks 11 and 21, reduced NADPH2 levels were observed in almost all examined groups. Co-administration of DEX and CVD negatively affected transferrin levels in DOX-treated rats but did not influence body weight changes. Ascites predominantly resulted from cardiac muscle dysfunction rather than liver-related effects. The study's findings, exploring the impact of DEX and CVD on DOX-induced cardiotoxicity, indicate a lack of scientific justification for advocating the combined use of these drugs at histological, biochemical, and molecular levels.

Machine-Learning-Guided Engineering of an NADH-Dependent 7ß-Hydroxysteroid Dehydrogenase for Economic Synthesis of Ursodeoxycholic Acid.

Enzymatic synthesis of ursodeoxycholic acid (UDCA) catalyzed by an NADH-dependent 7ß-hydroxysteroid dehydrogenase (7ß-HSDH) is more economic compared with an NADPH-dependent 7ß-HSDH when considering the much higher cost of NADP+/NADPH than that of NAD+/NADH. However, the poor catalytic performance of NADH-dependent 7ß-HSDH significantly limits its practical applications. Herein, machine-learning-guided protein engineering was performed on an NADH-dependent Rt7ß-HSDHM0 from Ruminococcus torques. We combined random forest, Gaussian Naïve Bayes classifier, and Gaussian process regression with limited experimental data, resulting in the best variant Rt7ß-HSDHM3 (R40I/R41K/F94Y/S196A/Y253F) with improvements in specific activity and half-life (40 °C) by 4.1-fold and 8.3-fold, respectively. The preparative biotransformation using a "two stage in one pot" sequential process coupled with Rt7ß-HSDHM3 exhibited a space-time yield (STY) of 192 g L-1 d-1, which is so far the highest productivity for the biosynthesis of UDCA from chenodeoxycholic acid (CDCA) with NAD+ as a cofactor. More importantly, the cost of raw materials for the enzymatic production of UDCA employing Rt7ß-HSDHM3 decreased by 22% in contrast to that of Rt7ß-HSDHM0, indicating the tremendous potential of the variant Rt7ß-HSDHM3 for more efficient and economic production of UDCA.

Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.

Protein cost minimization promotes the emergence of coenzyme redundancy.

SignificanceMetabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes [e.g., NAD(H) and NADP(H)] associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling and structural and sequence analysis indicates that coenzyme redundancy may not be essential for metabolism but could rather constitute an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems.

Assessment of Mitochondrial Membrane Potential and NADH Redox State in Acute Brain Slices.

Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron-glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.

A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry.

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of denovo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

Assessment of microvascular function in vivo using flow mediated skin fluorescence (FMSF) in patients with obstructive lung diseases: A preliminary study.

BACKGROUND: Cardiovascular diseases play an important role in the morbidity and mortality of patients with obstructive lung diseases. Impaired vascular endothelial function seems to be a key element linking obstructive lung disease and cardiovascular disease. Recently developed technique named flow mediated skin fluorescence (FMSF) is a novel, non-invasive tool to study microvascular function. METHODS: Total of 69 volunteers including 26 patients with chronic obstructive pulmonary disease (COPD), 23 patients with asthma and 20 healthy subjects underwent microvascular function assessments using FMSF. FMSF assessments were composed of measurements of reduced form of nicotinamide adenine dinucleotide (NADH) fluorescence intensity signal during brachial artery occlusion - ischemic response (IRmax) and immediately after release of occlusion - hyperemic response (HRmax). Associations of microvascular function with clinical and biochemical characteristics of studied subjects were also evaluated. RESULTS: The median value of IRmax was significantly lower in COPD subjects (2.4 [1.0-6.7] %) compared with healthy subjects (9.6 [3.7-13.5] %; p < 0.01). The mean value of HRmax was also significantly reduced in COPD subjects (9.7 (4.5) %) compared with both asthma subjects (12.1 (3.5) %; p < 0.05) and healthy control subjects (13.4 (2.9) %; p < 0.01). CONCLUSIONS: The FMSF technique makes it possible to identify impairments of the microvascular function in patients with COPD, but not in asthma patients. These exploratory findings require further validation in a larger patients cohort.

Assessment of the metabolism and morphology of the porcine cornea, lens and retina by 2-photon imaging.

Two-photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2-photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom-built 2-photon microscope for 5-dimensional microscopy with a near infrared broadband sub-15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.

Assessment of the low inhibitory specificity of oxamate, aminooxyacetate and dichloroacetate on cancer energy metabolism.

BACKGROUND: Exceedingly high therapeutic/experimental doses of metabolic drugs such as oxamate, aminooxyacetate (AOA) and dichloroacetate (DCA) are required to diminish growth, glycolysis and oxidative phosphorylation (OxPhos) of different cancer cells. To identify the mechanisms of action of these drugs on cancer energy metabolism, a systematic analysis of their specificities was undertaken. METHODS: Hepatocarcinoma AS-30D cells were treated with the inhibitors and glycolysis and OxPhos enzyme activities, metabolites and fluxes were analyzed. Kinetic modeling of glycolysis was used to identify the regulatory mechanisms. RESULTS: Oxamate (i) not only inhibited LDH, but also PYK and ENO activities inducing an increase in the cytosolic NAD(P)H, Fru1,6BP and DHAP levels in AS-30D cells; (ii) it slightly inhibited HPI, ALD and Glc6PDH; and (iii) it inhibited pyruvate-driven OxPhos in isolated heart mitochondria. AOA (i) strongly inhibited both AAT and AlaT, and 2-OGDH and glutamate-driven OxPhos; and (ii) moderately affected GAPDH and TPI. DCA slightly affected pyruvate-driven OxPhos and Glc6PDH. Kinetic modeling of cancer glycolysis revealed that oxamate inhibition of LDH, PYK and ENO was insufficient to achieve glycolysis flux inhibition. To do so, HK, HPI, TPI and GAPDH have to be also inhibited by the accumulated Fru1,6BP and DHAP induced by oxamate. CONCLUSION: Oxamate, AOA, and DCA are not specific drugs since they inhibit several enzymes/transporters of the glycolytic and OxPhos pathways through direct interaction or indirect mechanisms. GENERAL SIGNIFICANCE: These data explain why oxamate or AOA, through their multisite inhibitory actions on glycolysis or OxPhos, may be able to decrease the proliferation of cancer cells.

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis.

BACKGROUND: Analysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of this formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms. METHODS: To overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlo sampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined. RESULTS: The framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions. CONCLUSIONS: Our results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation. GENERAL SIGNIFICANCE: This framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.

An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

A Method of Accounting for Enzyme Costs in Flux Balance Analysis Reveals Alternative Pathways and Metabolite Stores in an Illuminated Arabidopsis Leaf.

Flux balance analysis of plant metabolism is an established method for predicting metabolic flux phenotypes and for exploring the way in which the plant metabolic network delivers specific outcomes in different cell types, tissues, and temporal phases. A recurring theme is the need to explore the flexibility of the network in meeting its objectives and, in particular, to establish the extent to which alternative pathways can contribute to achieving specific outcomes. Unfortunately, predictions from conventional flux balance analysis minimize the simultaneous operation of alternative pathways, but by introducing flux-weighting factors to allow for the variable intrinsic cost of supporting each flux, it is possible to activate different pathways in individual simulations and, thus, to explore alternative pathways by averaging thousands of simulations. This new method has been applied to a diel genome-scale model of Arabidopsis (Arabidopsis thaliana) leaf metabolism to explore the flexibility of the network in meeting the metabolic requirements of the leaf in the light. This identified alternative flux modes in the Calvin-Benson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the light-dependent contribution of alternative electron flow pathways and futile cycles in energy rebalancing. Notable features of the analysis include the light-dependent tradeoff between the use of carbohydrates and four-carbon organic acids as transitory storage forms and the way in which multiple pathways for the consumption of ATP and NADPH can contribute to the balancing of the requirements of photosynthetic metabolism with the energy available from photon capture.

An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis.

Three C4 acid decarboxylases, phosphoenolpyruvate carboxykinase (PEPCK), NADP-malic enzyme (NADP-ME), and NAD-malic enzyme (NAD-ME) were recruited from C3 plants to support C4 photosynthesis. In Poaceae, there are established lineages having PEPCK type species, and some NADP-ME lineages in which PEPCK contributes to C4. Besides family Poaceae, recently PEPCK has been reported to function in C4 photosynthesis in eudicot species including Cleome gynandra (Cleomaceae), Trianthema portulacastrum and Zaleya pentandra (Aizoaceae). We evaluated PEPCK by enzyme assay and western blots in representatives of Poaceae, Aizoaceae, Cleomaceae, and Chenopodiaceae compared to that in the PEPCK type C4 grass Spartina anglica. Eragrostis nutans was identified as the first NAD-ME type C4 grass having substantial amounts of PEPCK. In the eudicots, including C. gynandra, Cleome angustifolia, T. portulacastrum, Z. pentandra, and nine C4 members of family Chenopodiaceae (which has the most C4 species and diversity in forms among eudicot families), amounts of PEPCK were generally very low (barely detectable up to 4% of that in S. anglica). Based on these results, C4 species can be classified biochemically according to the dominant decarboxylase recruited for C4 function; and, Poaceae remains the only family in which PEPCK is known to have a significant role in C4 photosynthesis.

Assessment of testicular corticosterone biosynthesis in adult male rats.

Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.

High resolution in vivo 31P-MRS of the liver: potential advantages in the assessment of non-alcoholic fatty liver disease.

BACKGROUND: Biopsy remains the current gold-standard for assessing non-alcoholic fatty liver disease (NAFLD). To develop a non-invasive means of assessing the disease, 31P magnetic resonance spectroscopy (31P-MRS) has been explored, but the severe spectral overlaps and low signal-to-noise-ratio in 31P-MRS spectra at clinical field strength are clearly limiting factors. PURPOSE: To investigate potential advantages of high resolution in vivo 31P-MRS in assessing NAFLD. MATERIAL AND METHODS: The study was conducted at 9.4T in control and carbon tetrachloride (CCl4)-treated rats. Rats were divided according to histopathologic findings into a control group (n = 15), a non-alcoholic steatohepatitis group (n = 17), and a cirrhosis group (n = 12). Data were presented with different reference peaks that are commonly used for peak normalization such as total phosphorous signal, phosphomonoester + phosphodiester (PME + PDE), and nucleotide triphosphate (NTP). Then, multivariate analyses were performed. RESULTS: In all spectra PME and PDE were well resolved into phosphoethanolamine (PE) and phosphocholine (PC), and into glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC), respectively. Those MRS measures quantifiable only in highly resolved spectra had higher correlations with histology than those conventional MRS measures such as PME, PDE, and NTP. The optimized partial least-squares discriminant analysis (PLS-DA) model correctly classified 79% (22/28) of the rats in the training set and correctly predicted 69% (11/16) of the rats in the test set. CONCLUSION: PE, PC, GPE, GPC, and nicotinamide adenine dinucleotide phosphate (NADP) that can be separately quantifiable in highly resolved spectra may further improve the potential efficacy of 31P-MRS in the diagnosis of NAFLD.

The metabolic cost of flagellar motion in Pseudomonas putida KT2440.

Although the flagellar machinery of environmental bacteria endows cells with a phenomenal survival device, it also consumes much of the metabolic currency necessary for fuelling such a vigorous nano-motor. The physiological cost of flagella-related functions of the soil bacterium Pseudomonas putida KT2440 was examined and quantified through the deletion of a ≈ 70 kb DNA segment of the genome (≈ 1.1%), which includes relevant structural and regulatory genes in this micro-organism. The resulting strain lacked the protruding polar cords that define flagella in the wild-type P. putida strain and was unable of any swimming motility while showing a significant change in surface hydrophobicity. However, these deficiencies were otherwise concomitant with clear physiological advantages: rapid adaptation of the deleted strain to both glycolytic and gluconeogenic carbon sources, increased energy charge and, most remarkably, improved tolerance to oxidative stress, reflecting an increased NADPH/NADP(+) ratio. These qualities improve the endurance of non-flagellated cells to the metabolic fatigue associated with rapid growth in rich medium. Thus, flagellar motility represents the archetypal tradeoff involved in acquiring environmental advantages at the cost of a considerable metabolic burden.

A method for accounting for maintenance costs in flux balance analysis improves the prediction of plant cell metabolic phenotypes under stress conditions.

Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade-off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper-osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper-osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance.

Rapid and quantitative measurement of metabolic stability without chromatography or mass spectrometry.

Metabolic stability measurements are a critical component of preclinical drug development. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive and labor intensive. We have developed a general method to determine the metabolic stability of virtually any compound by quantifying cofactors in the mechanism of cytochrome P450 enzymes using fluorescence intensity measurements. While many previous studies have shown that simple measurements of cofactor depletion do not correlate with substrate conversion (i.e., metabolic stability) in P450 systems, the present work employs a reaction engineering approach to simplify the overall rate equation, thus allowing the accurate and quantitative determination of substrate depletion from simultaneous measurements of NADPH and oxygen depletion. This method combines the accuracy and generality of chromatography with the ease, throughput, and real-time capabilities of fluorescence.

An efficient and economical MTT assay for determining the antioxidant activity of plant natural product extracts and pure compounds.

Antioxidants scavenge free radicals, singlet oxygen, and electrons in cellular redox reactions. The yellow MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] is reduced to a purple formazan by mitochondrial enzymes. NADPH is the basis of established in vitro cell viability assays. An antioxidant assay has been developed utilizing the redox reaction between MTT and selected natural product extracts and purified compounds. This simple, fast, and inexpensive MTT antioxidant assay is comparable with the lipid peroxidation inhibitory assay and can be mechanized to achieve high throughput.

Assessment of the inactivation potential of desethylamiodarone on human CYP1A1.

Desethylamiodarone was reported to inactivate human CYP1A1. To assess this, two protocols were implemented employing dilution and non-dilution of the preincubation mixture. Inactivation studies performed with diluted preincubation mixtures showed no inactivation of CYP1A1 by desethylamiodarone. However there was evidence for a mixed competitive inhibition (competitive and the uncompetitive inhibition constants of 2.1 microM and 9.6 microM, respectively) of CYP1A1 by desethylamiodarone. NADPH addition and/or replenishment were found to be important factors in determining the control activity in inactivation studies.