Nicotinamide Adenine Dinucleotide Phosphate System Research Articles

CYP3A4/5 mediates the metabolic detoxification of humantenmine, a highly toxic alkaloid from Gelsemium elegans Benth.

Gelsemium elegans Benth., a well-known toxic herbal plant, is widely used to treat rheumatic arthritis, inflammation and other diseases. Gelsemium contains humantenmine (HMT), which is an important bioactive and toxic alkaloid. Cytochrome P450 enzymes (CYPs) play important roles in the elimination and detoxification of exogenous substances. This study aimed to investigate the roles of CYPs in the metabolism and detoxification of HMT. First, metabolic studies were performed in vitro by using human liver microsomes, selective chemical inhibitors and recombinant human CYPs. Results indicated that four metabolites, including hydroxylation and oxidation metabolites, were found in human liver microsomes and identified based on their high-resolution mass spectrum. The isoform responsible for HMT metabolism was mainly CYP3A4/5. Second, the toxicity of HMT on L02 cells in the presence of the nicotinamide adenine dinucleotide phosphate system (NADPH) was significantly less than that without NADPH system. A CYP3A4/5 activity inhibition model was established by intraperitoneally injecting ketoconazole in mice and used to evaluate the role of CYP3A4/5 in HMT detoxification. In this model, the 14-day survival rate of the mice decreased to 17% after they were intragastrically treated with HMT, along with hepatic injury and increasing alanine aminotransferase (ALT) /aspartate aminotransferase (AST)levels. Overall, CYP3A4/5 mediated the metabolism and detoxification of HMT.

Interactions of the Antimalarial Drug Methylene Blue with Methemoglobin and Heme Targets inPlasmodium falciparum: A Physico-Biochemical Study

Resistance of Plasmodium falciparum to drugs has led to renewed interest of redox-active methylene blue (MB) for which no resistance has been reported so far. Moreover, MB displays unique interactions with glutathione reductase (GR). However, the mechanisms of action/interaction with potential targets of MB are yet to be elucidated. Our physico-biochemical study on MB and relevant hematin-containing targets was performed under quasi-physiological conditions. The water deprotonation of the Fe(III)protoporphyrin dimer, the major building block of β-hematin, was studied. At pH 6, the predominant dimer possesses water coordinated to both metals. Below pH 6, spontaneous precipitation of β-hematin occurred reminiscent of hemozoin biomineralization at pH 5.0-5.5 in the food vacuole of the malarial parasite. MB also forms dimers (K(Dim)=6800 M(-1)) and firmly binds to hematin in a 2:1 hematin:MB sandwich complex (K(D)=3.16 μM). MB bioactivation catalyzed by GR induces efficient methemoglobin(Fe(III)) [metHb(Fe(III))] reduction to hemoglobin(Fe(II)). The reduction rate, mediated by leucomethylene blue (LMB), was determined (k(metHb)(red)=991 M(-1)·s(-1)) in an assay coupled to the GR/reduced form of nicotinamide adenine dinucleotide phosphate system. Our work provides new insights into the understanding of (i) how MB interacts with hematin-containing targets, (ii) other relevant MB properties in corroboration with the distribution of the three major LMB species as a function of pH, and (iii) how this redox-active cycler induces efficient catalytic reduction of metHb(Fe(III)) to hemoglobin(Fe(II)) mediated by oxidoreductases. These physico-biochemical parameters of MB open promising perspectives for the interpretation of the pharmacology and pathophysiology of malaria and possibly new routes for antimalarial drug development.

Spectrofluorimetric determination of trace amount of coenzyme II using ciprofloxacin–terbium complex as a fluorescent probe

A new spectrofluorimetric method was developed for the determination of trace amount of nicotinamide adenine dinucleotide phosphate (NADP). Using terbium ion (Tb 3+)–ciprofloxacin (CIP) complex as a fluorescent probe, in the buffer solution of pH = 9.00 , NADP can remarkably enhance the fluorescence intensity of the Tb 3+–CIP complex at λ = 545 nm and the enhanced fluorescence intensity of Tb 3+ ion is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 4.9×10 −7−3.7×10 −6 mol L −1 with detection limit of 1.3×10 −7 mol L −1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Tb 3+-CIP system and the Tb 3+–CIP–NADP system have been also discussed.

Fluorimetric study of the interaction between nicotinamide adenine dinucleotide phosphate and tetracycline–europium complex and its application

A new spectrofluorimetric method was developed for the determination of trace amount of nicotinamide adenine dinucleotide phosphate (NADP). Using europium (Eu 3+)–tetracycline (TC) complex as a fluorescent probe, in the buffer solution of pH 7.60. NADP can remarkably enhance the fluorescence intensity of the Eu 3+–TC complex at λ = 612 nm and the enhanced fluorescence intensity of Eu 3+ ion is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 4.4 × 10 −7 to 2.2 × 10 −6 mol l −1 with detection limit of 6.9 × 10 −8 mol l −1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples and in serum samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Eu 3+–TC system and the Eu 3+–TC–NADP system have been also discussed.

The calculation of the cytoplasmic free [NADP +]/[NADPH] ratio in brain: Effect of electroconvulsive seizure

This study has investigated the feasibility of calculating the cytoplasmic free [NADP +]/[NADPH] ratio in rat brain. The time course of the change in the substrate ratios of the malate dehydrogenase (decar☐ylating) [E.C. 1.1.1.40], NADP +-isocitrate dehydrogenase (decar☐ylating) [E.C. 1.1.1.42] and 6-phosphogluconate dehydrogenase (decar☐ylating) [E.C. 1.1.1.44] reactions was followed for up to 10 min after a single, unmodified electroconvulsive seizure. From the results it has been concluded that during periods of low flux, the direction and magnitude of the change in the cytoplasmic free [NADP +]/[NADPH] ratio can, in fact, be reasonably determined even though ther is some uncertainty in the absolute value of the ratio itself. It is recommended that reliance not be placed on a single enzyme system but that one or both of the other systems also be observed under a given experimental condition to increase confidence in the determination. The results also demonstrate that seizure and anoxia have a far lesser effect on the cytoplasmic free [NADP +]/[NADPH] ratio than on the free [NAD +]/[NADH] ratio in the same compartment. These results suggest that the pathways using the nicotinamide-adenine dinucleotide phosphate system are relatively protected from the rapid fluctuations that seizure and anoxia can produce.

Use of tetrazolium salts for electron transport studies in meningopneumonitis. II. Reduced nicotinamide adenine dinucleotide phosphate system.

Allen, Emma G. (Downstate Medical School, State University of New York, Brooklyn). Use of tetrazolium salts for electron transport studies in meningopneumonitis. II. Reduced nicotinamide adenine dinucleotide phosphate system. J. Bacteriol. 92:1041-1046. 1966.-The conditions of electron transfer from nicotinamide adenine dinucleotide phosphate in meningopneumonitis (MP) are described and compared with electron transfer from nicotinamide adenine dinucleotide by this organism. The observations suggest that, in either system, there may be more than one pathway of electron flow, and that these pathways differ from those in normal membrane particulates. It was also found that after trypsin treatment, particulates from pools of normal allantoic fluids and membranes retain the normal characteristics, whereas those from pools of MP-infected fluids and membranes assume the characteristics of MP particles from fluids.