Bioadsorption, bioaccumulation and biodegradation of antibiotics by algae and their association with algal physiological state and antibiotic physicochemical properties.

Bioadsorption, bioaccumulation and biodegradation processes in algae, play an important role in the biomagnification of antibiotics, or other organic pollutants, in aquatic food chains. In this study, the bioadsorption, bioaccumulation and biodegradation of norfloxacin [NFX], sulfamethazine [SMZ] and roxithromycin [RTM]) is investigated using a series of culture experiments. Chlorella vulgaris was exposed to these antibiotics with incubation periods of 24, 72, 120 and 168 h. Results show the bioadsorption concentration of antibiotics in extracellular matter increases with increasing alkaline phosphatase activity (AKP/ALP). The bioaccumulation concentrations of NFX, SMZ and RTM within cells significantly increase after early exposure, and subsequently decrease. There is a significant positive antibiotics correlation to superoxide dismutase (SOD), the photosynthetic electron transport rate (ETR) and maximum fluorescence after dark adaptation (Fv/Fm), while showing a negative correlation to malondialdehyde (MDA). The biodegradation percentages (Pb) of NFX, SMZ and RTM range from 39.3 - 97.2, 41.3 - 90.5, and 9.3 - 99.9, respectively, and significantly increase with increasing Fv/Fm, density and chlorophyll-a. The accumulation of antibiotics in extracellular and intracellular substances of C. vulgaris is affected by antibiotic biodegradation processes associated with cell physiological state. The results succinctly explain relationships between algal growth during antibiotics exposure and the bioadsorption and bioaccumulation of these antibiotics in cell walls and cell matter. The findings draw an insightful understanding of the accumulation of antibiotics in algae and provide a scientific basis for the better utilization of algae treatment technology in antibiotic contaminated wastewaters. Under low dose exposures, the biomagnification of antibiotics in algae is affected by bioadsorption, bioaccumulation and biodegradation.

Influence of metabolism and microbiology on organic micropollutants biotransformation in anoxic heterotrophic reactors.

There is scarce information about the biotransformation of organic micropollutants (OMPs) under anoxic conditions. In this study, a heterotrophic denitrifying bioreactor was set up to study the fate of several OMPs from metabolic and microbiological points of view. Primary metabolic activity was increased by adding progressively higher nitrogen loading rates during the operation (from 0.075 to 0.4 g N-NO3- L-1 d-1), which resulted in an important shift in the microbial population from a specialized biomass to a more diverse community. Such a change provoked a significant increase in the removal efficiency of erythromycin (ERY), roxithromycin (ROX) and bisphenol-A (BPA), and some bacterial taxa, such as Rhodoplanes, were identified as possible indicators related to the biodegradation of these compounds. The increasing primary metabolic activity in the reactor did not enhance the OMP-specific removal rates, suggesting that the bacterial composition is more influential than cometabolism.

Aged microplastics change the toxicological mechanism of roxithromycin on Carassius auratus: Size-dependent interaction and potential long-term effects.

Size effects of microplastics have received extensive attention for their influence on other pollutants and harm to organisms. In this study, we investigated the uptake, elimination, tissue distribution and potential toxicity mechanism of roxithromycin (ROX) in the presence of 0.5, 5 and 50 µm of aged microplastics (AMPs) in Carassius auratus. The results showed that AMPs promoted the ROX bioaccumulation of various tissues in a size-dependent manner. AMPs and ROX significantly induced superoxide dismutase and catalase activities of liver and gut, and inhibited acetylcholinesterase activities of brain. The coexistence of smaller AMPs exacerbated pathological abnormalities in liver, gill and brain induced by ROX, while larger AMPs caused more intestinal damage. Moreover, high-throughput 16S rRNA gene sequencing indicated that the abundance of Proteobacteria in 0.5 µm AMPs and ROX joint treatments and Firmicutes and Bacteroidota in 50 µm AMPs and ROX joint treatments were significantly raised (p < 0.05). Metabolomics revealed that AMPs and ROX had a size-dependent long-term effect on gut microbial metabolites, which was mainly related to galactose metabolism, amino acid metabolism and primary bile acid biosynthesis pathways after a 7-day elimination, respectively. These results provide important insights into the relationship between the size effect of AMPs and interaction mechanism of AMPs and coexisting pollutants on aquatic organisms.

Roxithromycin inhibits compound 48/80-induced pseudo-allergy via the MrgprX2 pathway both in vitro and in vivo.

Roxithromycin (ROX) is a macrolide antibiotic with a variety of immunological effects. Mast cells (MCs) play a key role in host defense, mediating hypersensitivity and pseudo-allergic reactions. Mas-related G protein-coupled receptor X2 (MrgprX2) is the main receptor related to pseudo-allergy. In this study, we investigated the anti-pseudo-allergy effect of ROX and its underlying mechanism. The effects of ROX on passive cutaneous anaphylaxis (PCA) and active systemic allergy were examined, degranulation, Ca2+ influx, and cytokine release were studied in vivo and in vitro. Interactions between ROX and MrgprX2 protein were also detected through surface plasmon resonance. The PCA and active systemic allergy induced by compound 48/80 were inhibited by ROX. An intermolecular interaction was detected between the ROX and MrgprX2 protein. In conclusion, ROX could inhibit pseudo-allergic reactions, and this effect involves the Ca2+/PLC/IP3 pathway of MrgprX2. This study provides new insight into the anti-pseudo-allergy effects of ROX.

Interactive effects of polystyrene microplastics and roxithromycin on bioaccumulation and biochemical status in the freshwater fish red tilapia (Oreochromis niloticus).

There are hundreds of thousands metric tons of microplastics (MPs) present in aquatic environments. The MPs coexist with other pollutants in water bodies, such as pharmaceuticals, and may carry and transfer them into aquatic organisms, consequently causing unpredictable ecological risks. The purpose of the present study was to evaluate the effect of the presence of polystyrene microplastics (PS-MPs) on the distribution and bioaccumulation of roxithromycin (ROX) in freshwater fish red tilapia (Oreochromis niloticus) as well as their interactive biochemical effects in red tilapia. PS-MPs were found to enhance the bioaccumulation of ROX in fish tissues compared to ROX-alone exposure. In the treatment of PS-MPs (100 µg L-1) combined with ROX (50 µg L-1), the highest concentrations of ROX reached 39,672.9 ±â€¯6311.4, 1767.9 ±â€¯277.8, 2907.5 ±â€¯225.0, and 4307.1 ±â€¯186.5 µg kg-1 in gut, gills, brain, and liver, respectively. Furthermore, compared to the ROX alone, the neurotoxicity caused by ROX was alleviated due to the presence of MPs after 14 d of exposure. The activities of cytochrome P450 (CYP) enzymes [7-ethoxyresorufin O-deethylase (EROD) and 7-benzyloxy-4-trifluoromethyl-coumarin O-dibenzyloxylase (BFCOD)] in fish livers exposed to all co-exposure treatments exhibited great variability compared to ROX alone after 14 d of exposure, suggesting that the presence of MPs may affect the metabolism of ROX in tilapia. Compared with ROX alone, the superoxide dismutase (SOD) activity increased significantly, and malondialdehyde (MDA) contents decreased in the co-exposure treatments, showing that oxidative damage in situations of co-exposure to MPs and ROX was mitigated in fish livers after 14 d of exposure. Collectively, the presence of MPs could affect the fate and toxicity of other organic pollutants in fish. The results emphasize the importance to study the interactions between MPs and other organic pollutants in aquatic environments.

Preparation, characterization and in vitro release study of drug-loaded sodium carboxy-methylcellulose/chitosan composite sponge.

A sodium carboxy-methylcellulose (CMC)/chitosan (CS) composite sponge as drug carrier was prepared, and its structure and functions were investigated. Samples with different CMC/chitosan ratios and under different pH conditions were synthesized via a freeze-drying method. The microstructure of the dried sponges was analyzed by Scanning Electron Microscope (SEM). Molecule interactions between polymers were confirmed by Fourier transform infrared (FTIR) spectra and Thermal gravimetric analyze (TGA). The swelling degree, weight loss, in vitro drug release behavior and antibacterial property of the sponges were determined as well. The results showed that the CMC/chitosan ratio and the pH value significantly affected the appearance of the blending solution and the microstructure of the final product, and also affected the sponge's degradation behavior, drug-loading capacity and the antibacterial activity. Gentamicin (GEN) as a hydrophilic model drug was remarkably superior to the other two hydrophobic drugs, ibuprofen (IBU) and roxithromycin (ROX), with respect to in vitro releasing. Moreover, higher CMC content and lower pH value of the sponge were confirmed to lead a larger loading for GEN. The bacteriostatic experiment showed a strong antimicrobial ability of GEN-loaded sponges on inhibiting Escherichia coli.

Interactive effects of selected pharmaceutical mixtures on bioaccumulation and biochemical status in crucian carp (Carassius auratus).

The aim of this study was to evaluate the interactive effects of fluoxetine (FLU), roxithromycin (ROX) and propranolol (PRP) on the bioaccumulation and biochemical responses in the crucian carp Carassius auratus. After 7 days of binary exposure (ROX + FLU and PRP + FLU), the addition of waterborne FLU at nominal concentrations of 4, 20 and 100 µg L(-1) significantly increased the accumulation of ROX and PRP in fish livers in most cases, although elevated ROX and PRP bioaccumulation levels were not observed in muscles or gills. The inductive response of 7-ethoxyresorufin O-deethylase (EROD) to PRP and that of 7-benzyloxy-4-trifluoromethyl-coumarin O-dibenzyloxylase (BFCOD) to ROX were inhibited by the co-administration of FLU at all tested concentrations. Correspondingly, marked inhibition of CYP1A and CYP3A mRNA expression levels was observed in the livers of fish co-treated with FLU + PRP and FLU + ROX relative to their PRP- and ROX-only counterparts, respectively. In addition, as reflected by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, co-exposure to ROX + FLU and PRP + FLU seemed to induce stronger antioxidant responses than single pharmaceutical exposure in fish livers. This work indicated that the interactive effects of pharmaceutical mixtures could lead to perturbations in the bioaccumulation and biochemical responses in fish.

Bioconcentration, metabolism, and biomarker responses in freshwater fish Carassius auratus exposed to roxithromycin.

To investigate the distribution, bioconcentration, metabolism, and biomarker responses of macrolide antibiotic roxithromycin (ROX) in fish, crucian carp (Carassius auratus) were exposed to various concentrations of ROX (4, 20, and 100µgL(-1)) for 20d. The ROX content in different tissues was quantified using UPLC/MS/MS. The liver exhibited the highest ROX concentration followed by the bile, gills, and muscle tissues. After 15d of exposure to different concentrations of ROX, the bioconcentration factors were 2.15-38.0 in the liver, 0.950-20.7 in the bile, 0.0506-19.7 in the gill, and 0.0439-13.8 in the muscle; these results were comparable to the estimated BCF values. The metabolites formed in the bile were identified based on metabolic identification in human bile. Additionally, the biomarkers, including acetylcholinesterase in the brain, as well as 7-ethoxyresorufin O-deethylase and superoxide dismutase in the liver changed significantly after 5, 10, 15, and 20d of exposure (P<0.05). Our results suggest that ROX can accumulate and be metabolized in fish; therefore, interactions between ROX or its metabolites and the biological systems may induce biochemical disturbances in fish.

Effects of roxithromycin on ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the rhizosphere of wheat.

In a pot-cultural experiment, the impact of the antibiotic roxithromycin (ROX) addition was assessed on the diversities of microbial structure and function communities, especially involved in ammonia and nitrite oxidation in wheat rhizosphere soil with and without the addition of earthworms. The abundances of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total bacteria were surveyed by the quantitative PCR. The quantities of total bacteria, AOB, and NOB with earthworms were higher than those without earthworms because of the synergistic effect. ROX inhibited the growth of AOB in all treatments, although the quantities of AOB were in a light increase in medium and heavy polluted treatments compared with that in the light polluted treatments. Different from AOB, the quantities of NOB were lowest in light polluted treatments, but the quantities of NOB were rapidly increased in medium and heavy polluted treatments compared with that in the control. These results indicated that the application of ROX principally had a negative effect on nitrification performance by affecting the abundances and relative ratios of both AOB and NOB in soil communities, which affected the N cycle in an agricultural ecosystem. According to the metabolic diversities evaluated by the biologic assay, the tendency of metabolic diversities was quite contrary to the quantities of NOB in all treatments and showed the contrast growing relation of autotrophic and heterotrophic bacteria under ROX pollution pressure in agricultural ecosystems.

Molecular insights into 14-membered macrolides using the MM-PBSA method.

Erythromycin A and roxithromycin are clinically important macrolide antibiotics that selectively act on the bacterial 50S large ribosomal subunit to inhibit bacteria's protein elongation process by blocking the exit tunnel for the nascent peptide away from ribosome. The detailed molecular mechanism of macrolide binding is yet to be elucidated as it is currently known to the most general idea only. In this study, molecular dynamics (MD) simulation was employed to study their interaction at the molecular level, and the binding free energies for both systems were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The calculated binding free energies for both systems were slightly overestimated compared to the experimental values, but individual energy terms enabled better understanding in the binding for both systems. Decomposition of results into residue basis was able to show the contribution of each residue at the binding pocket toward the binding affinity of macrolides and hence identified several key interacting residues that were in agreement with previous experimental and computational data. Results also indicated the contributions from van der Waals are more important and significant than electrostatic contribution in the binding of macrolides to the binding pocket. The findings from this study are expected to contribute to the understanding of a detailed mechanism of action in a quantitative matter and thus assisting in the development of a safer macrolide antibiotic.

pH-dependent geometric isomerization of roxithromycin in simulated gastrointestinal fluids and in rats.

The biotransformation of roxithromycin in simulated gastrointestinal fluids at 37 degrees C and in rats was investigated by using liquid chromatography-tandem mass spectrometry. Roxithromycin degraded to its Z-isomer and decladinose derivative in simulated gastrointestinal fluids in vitro at pH

[Metabolism of roxithromycin in dogs].

AIM: To investigate the metabolic profile of roxithromycin in dogs and the effects of oral and intravenous administrations on the metabolism of roxithromycin. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MSn) was used for separation and analysis of roxithromycin and its metabolites in dog bile after an oral dose or intravenous dose of roxithromycin. The metabolites were identified by comparisons of their mass spectra and LC behaviors with the references. RESULTS: Totally 13 metabolites were detected in dog bile, including N-demethylated derivatives, N, N-didemethylated derivatives, O-dealkylether derivatives, decladinose derivatives, and the geometric isomers of parent drug and its metabolites. CONCLUSION: Roxithromycin underwent 4 metabolic pathways in which geometric isomerization and decladinose metabolism were found to be markedly different between the two administration routes.

Metabolism of roxithromycin in phenobarbital-treated rat liver microsomes.

AIM: To investigate the metabolism of roxithromycin (RXM) in rat liver microsomes and the possible effects of RXM and its metabolites on cytochrome P-450 (CYP450). METHODS: Liver microsomes of Wistar rats, induced by phenobarbital, were prepared using ultracentrifuge method. RXM in vitro metabolism was stu died with the microsome incubation. The metabolites were separated and assayed by li quid chromatography-tandem mass spectrometry (LC-MSn), and were further identified by comparison of their mass spectra and LC behavior to synthesized references. RESULTS: N-Mono- and N-di-demethyl metabolites a s well as O-dealkylated metabolite (erythromycin oxime) were detected in microsomal incubates. RXM and its metabolites expressed weak potency to form inactive complexes with CYP450. CONCLUSION: N-Demethylation and oxime ether side chain O- dealkylation are main biotransformation pathways of RXM in phenobarbital-treated rat liver microsomes. Both routes were found to be NADPH-dependent. RXM and its metabolites showed weak inhibitory effects on CYP450.

Demethylation metabolism of roxithromycin in humans and rats.

AIM: To investigate the demethylated metabolites of roxithromycin (RXM) in humans and rats, and to study the antibiotic activity of these metabolites in vitro. METHODS: The demethylated metabolites of RXM in humans and in rats were identified by liquid chromatography-mass spectrometry (LC-MS), and the in vitro antibiotic activities of them against three standard strains were also studied compared with those of the parent drug and some other metabolites of RXM. RESULTS: O-Demethylation of RXM was one of the main metabolic routes of RXM in humans, whereas N-demethylation metabolism was more predominant in rats. O-Demethyl-RXM appeared to be equally effective with RXM. CONCLUSION: The O-demethyl-RXM was an active metabolite in humans, and there were some species differences in RXM demethylation metabolism between humans and rats.

Applications of liquid chromatography-tandem mass spectrometry in drug and biomedical analyses.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the major tools for bioanalytical works. These instruments can be used for quantitative determination of plasma samples in pharmacokinetic studies. LC-MS/MS techniques frequently provide specific, selective and sensitive quantitative results often with reduced sample preparation and analysis time relative to other commonly employed techniques. METHODS: The applications of these instruments include analysis of nine corticosteroids illegally adulterated in traditional Chinese medicines, identification of drug metabolites of roxithromycin, propafenone and some glucuronide conjugates and the quantitative determination of plasma samples containing amlodipine, nitrendipine, flupirtine and their major metabolites. CONCLUSIONS: LCQ is ideally suited for the rapid identification of metabolites because it provides on-line LC/MS(n) capability, enabling multiple MS stages to obtain the maximum amount of structural information, while TSQ provides excellent precise and accurate results for quantitative assays at very low detection limits.

Effect of proinflammatory cytokines on the interplay between roxithromycin, HMR 3647, or HMR 3004 and human polymorphonuclear neutrophils.

Cytokines, the hallmarks of infectious and inflammatory diseases, modify phagocyte activities and thus may interfere with the immunomodulating properties of antibacterial agents. We have investigated whether various proinflammatory cytokines (interleukin 1 [IL-1], IL-6, IL-8, gamma interferon, tumor necrosis factor alpha [TNF-alpha], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) modify two macrolide properties, i.e., inhibition of oxidant production by polymorphonuclear neutrophils (PMN) and cellular uptake. Roxithromycin and two ketolides, HMR 3647 and HMR 3004, were chosen as the test agents. TNF-alpha and GM-CSF (but not the other cytokines) decreased the inhibitory effect of HMR 3647 only on oxidant production by PMN. Fifty percent inhibitory concentrations were, however, in the same range in control and cytokine-treated cells (about 60 to 70 microgram/ml), suggesting that HMR 3647 acts downstream of the priming effect of cytokines. In contrast, the impairment of oxidant production by roxithromycin and HMR 3004 was unchanged (or increased) in cytokine-treated cells. This result suggests that HMR 3004 (the strongest inhibitory drug, likely owing to its quinoline side chain) and roxithromycin act on a cellular target upstream of cytokine action. In addition, TNF-alpha and GM-CSF significantly (albeit moderately) impaired (by about 20%) the uptake of the three molecules by PMN. The inhibitory effect of these two cytokines seems to be related to activation of the p38 mitogen-activated protein kinase. Our data also illuminate the mechanism underlying macrolide uptake: protein kinase A- and tyrosine kinase-dependent phosphorylation seems to be necessary for optimal uptake, while protein kinase C activation impairs it. The relevance of our data to the clinical setting requires further investigations, owing to the complexity of the cytokine cascade during infection and inflammation.

Formation in vitro of an inhibitory cytochrome P450 x Fe2+-metabolite complex with roxithromycin and its decladinosyl, O-dealkyl and N-demethyl metabolites in rat liver microsomes.

1. Roxithromycin and its major metabolites found in rat and human urine, namely the decladinosyl derivative (M1), O-dealkyl derivative (M2) and N-demethyl derivative (M3), were incubated with rat liver microsomes and formation of an inhibitory cytochrome P450 (CYP)-metabolite complex and of formaldehyde (measurement of N-demethylation) were determined in vitro. Troleandomycin and erythromycin were also used for comparison. 2. Dexamethasone very significantly induced the microsomal N-demethylations of these macrolide antibiotics. The order of magnitude for the Vmax/Km ratio of N-demethylations by liver microsomes from dexamethasone-treated rats was troleandomycin > erythromycin = M2 > roxithromycin > M3, M1. 3. Formation of an inhibitory P450 x Fe2+-metabolite complex was detected on incubation of these macrolide antibiotics with rat liver microsomes in the presence of an NADPH-generating system and the order of maximum complex formation was troleandomycin > erythromycin > M2 > roxithromycin > M3 > M1. 4. Troleandomycin, erythromycin and M2 inhibited CYP3A-dependent testosterone 6beta-hydroxylation catalysed by liver microsomes from the dexamethasone-treated rat by 54, 33 and 23%, respectively, but roxithromycin, M3 and M1 were very weak by comparison. In the untreated rat, only testosterone 6beta-hydroxylation, but not testosterone 16alpha- and 2alpha-hydroxylation and androstenedione formation, activities were inhibited, indicating that inhibitory actions of these antibiotics are specific for CYP3A enzymes in liver microsomes. 5. These results support the view that formation of an inhibitory P450-metabolite complex is prerequisite for the inhibition of CYP3A-dependent substrate oxidations by rat liver microsomes and that M2 (and M3, to a lesser extent) may be the active metabolite that can form an inhibitory P450-metabolite complex by CYP3A enzyme(s).

Metabolism of roxithromycin in the isolated perfused rat liver.

Roxithromycin is a macrolide antibiotic with high clinical potency. N-Demethylation is considered to be one of the main pathways of roxithromycin metabolism in rats. We have studied the hepatic metabolism of roxithromycin in the isolated perfused rat liver. After addition of roxithromycin (30 microM) to the perfusion medium the parent compound and one major metabolite were detected in bile by high-performance liquid chromatography. The metabolite was identified as monodesmethylated roxithromycin by mass spectrometric analysis. Onset of biliary excretion of native roxithromycin was fast, reaching a maximum (130.52 +/- 43.88 pmol g(-1) min(-1)) after only 10 min, whereas excretion of the metabolite was delayed (maximum 75.83 +/- 11.92 pmol g(-1) min(-1) at 30 min). The cumulative excretion of roxithromycin and its metabolite into bile during the 60 min of application amounted to only 1.09 +/- 0.30 and 0.64 +/- 0.22% of the roxithromycin cleared from the perfusate during the same time. The liver content was 0.48 micromol (g liver)(-1), indicating high retention within the organ. No release of the metabolite into the perfusate was detected. In conclusion, this study has demonstrated the importance of phase-I metabolism for the biliary excretion of roxithromycin in rat liver. These findings might be predictive of roxithromycin biotransformation and biliary excretion in man.

Solution conformation of methylated macrolide antibiotics roxithromycin and erythromycin using NMR and molecular modelling. Ribosome-bound conformation determined by TRNOE and formation of cytochrome P450-metabolite complex.

Conformational study of methylated derivatives of macrolide antibiotics roxithromycin (6-OMe-roxithromycin and 6,11-OMe-roxithromycin) has been achieved by NMR in solution and molecular dynamics (MD) simulations and compared to 6-OMe-erythromycin (clarithromycin). A complete conformational study by NMR has been led by determination of homonuclear coupling constants and NOEs. Heteronuclear 1H-13C coupling constants were also measured to investigate the orientation of the sugar moieties with respect to the erythronolide. MD simulations were performed using the crystallographic coordinates as the starting conformation. For each compound, experimental results were compared to calculated conformations in order to identify eventual conformational equilibrium in solution. It is shown that the effect of the methylation is opposite for roxithromycin compared to erythromycin especially on motional properties as the roxithromycin derivatives gain in mobility while the erythromycin derivatives behaves as a more restrained molecule. The study of macrolide-ribosome interactions has been investigated using transferred NOESY 1H NMR experiments and the conformations weakly bound to bacterial ribosomes were determined. Biological interactions of these compounds with membranar liver protein cytochrome P450 was also discussed with regard to their structural properties.

Transferred nuclear Overhauser effect study of macrolide-ribosome interactions: correlation between antibiotic activities and bound conformations.

The study of macrolide-ribosome interactions has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). A new medically important macrolide antibiotic, roxithromycin, with the replacement of the 9-keto group in erythromycin by a 9-oxime chain, was studied in the complex state with the bacterial ribosome. Analysis of transferred nuclear Overhauser effect (TRNOE) experiment resulted in a set of constraints for all protons pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrate in solution indicate that only one specific conformation is preferred in the bound state while in the free state the sugar ring moities were relatively disordered. The bioactive macrolide antibiotics studied roxithromycin and erythromycin which displayed a strong NMR response, are metabolized in RU39001 and erythralosamine respectively which do not retain antimicrobial activity. The inactive major metabolites were used to define if TRNOEs observation may be characteristic of a biological activity. These control experiments gave essentially blank TRNOESY spectra. This study shows that Mg2+ does not play a direct role for the low affinity binding site studied by TRNOE what is in agreement with an hypothesis of two distinct binding levels, with a low affinity binding level necessary for the tight binding one.