Metabolites formed after incubation

Table II. Concentrations and enantiomeric compositions of metabolites formed after incubation of pineapple slices...

The distribution of metabolites obtained after incubation of pineapple slices with keto acids and keto esters, potential precursors of the corresponding hydroxy compounds, is summarized in Table II. The metabolization steps comprise esterification, reduction to hydroxy compounds, formation of acetoxy esters, and cyclization to the corresponding lactones. Metabolization rate and distribution of formed products strongly depend on the structures of the precursors. The detection of these metabolites proves the enzymatic capability of pineapple tissue to catalyze these conversions, an aspect which might be interesting for future use of pineapple tissue cultures in the production of chiral compounds. 

Huffman in 1942 expressed the view that 16-oxoestrone is not an end product of estrogen metabolism. In fact, after injection of 16-oxoestrone into man, estriol (Stimmel et al., 1948, 1950) and 16-epiestriol (Stimmel, 1958) were detected in urine. 16-Oxoestradiol-17 3 was apparently formed as an intermediate (Stimmel, 1958). That 16-oxoestrone is a key substance in the metabolism of the 16-substituted estrogens has been demonstrated by in vitro experiments (Breuer et al., 1959c). The metabolites found after incubation of 16-oxoe.strone with human liver slices were 16a-hydroxy-estrone, 16 8-hydroxyestronc, 16-oxoestradiol-l7 8, estriol, 16-epiestriol,... 

Fig. 1. Radiochromatogram of metabolites formed after 18 2 incubation with leaf homogenate in presence of 1 mM NADPH.

Metabolites formed during the decolourization of the azo dye Reactive red 22 by Pseudomonas luteola were separated and identified by HPLC-DAD and HPLC-MS. The chemical structures of Reactive red 22 (3-amino-4-methoxyphcnyl-/fhydroxyl-sulphonc sulphonic acid ester) and its decomposition products are shown in Fig. 3.92. RP-HPLC measurements were carried out in an ODS column using an isocratic elution of 50 per cent methanol, 0.4 per cent Na2HP04 and 49.6 per cent water. The flow rate was 0.5 ml/min, and intermediates were detected at 254 nm. The analytes of interest were collected and submitted to MS. RP-HPLC profiles of metabolites after various incubation periods are shown in Fig. 3.93. It was concluded from the chromatographic data that the decomposition process involves the breakdown of the azo bond resulting in two aromatic amines [154],... 

The administration of TNT to laboratory animals leads to the excretion of 4-NHOH-DNT, 2-NH2-DNT, and 4-NH2-DNT in the urine [59], and to the formation of covalent adducts with microsomal liver and kidney proteins, hemoglobin, and other blood proteins [60], The acid hydrolysis of adducts yielded mainly 2-NH2-DNT (2-ADNT) and 4-NH2-DNT (4-ADNT). Incubation of rat liver microsomes with TNT and NADPH under aerobic conditions resulted in the formation of NH2-DNTs and the transient metabolite 4-NHOH-DNT [57], The formation of covalent protein adducts with TNT metabolites was enhanced by the presence of 02 and decreased by GSH. This is consistent with the scheme of the TNT adduct formation with the central role of the nitroso metabolite (NO-DNT) reaction with protein or nonprotein thiols (RSH Equation 9.11) [57], The acid hydrolysis of the sulfinamide adduct (RS(0)-NH-DNT) formed after the rearrangement of the semimercaptal (RS-N(OH)-DNT Equation 9.12) will yield NH2-DNT. The mixture of NHOH-DNTs inhibits bacterial glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase more efficiently than TNT [61]. This was attributed to the covalent modification of protein -SH groups. 

Quick primary screens for CYP inhibition can fail to identify time-dependent inhibitors (TDIs) as noted in Box 10.2. These are not at all uncommon. To identify TDIs one sample of the test compound is incubated with microsomes or an rCYP without NADPH, while a second one otherwise identical, but containing this necessary CYP co-factor, is prepared. After 30 min the substrate and NADPH (now needed for metabolism to proceed) are added to each, and the amount of metabolite formed is determined for each sample. Time-dependent inhibitors will... 

In 1957, Mueller and Rumney showed that, when estradiol-17 9-76-C was incubated under aerobic conditions with mouse liver microsomes and NADPH2, a number of radioactive products more polar than estradiol-17/3 were formed. One of the products proved to be identical with 6-0x0-estradiol-17/3, whereas the two remaining phenolic substances were identified as 6 -hydroxyestrone and 6 j3 -hydroxycstradiol-17/9. Breuer et al. (1959a) next detected the same metabolites after incubation of estradiol-17/3 or estrone with rat liver slices. When these experiments were carried out, the absolute configuration of the 6-hydroxy group in the 6-hydroxy-estradiol-17/3 which served as a reference compound was unknown. The reference sample of 6-hydroxyestradiol-17/3 had been obtained by boro-hydride reduction of 6-oxoestradiol-17/8 (Longwell and Wintersteincr,... 

Fig. 11. HPLC traces of metabolites formed from 1- (A), 3- (B), and 7-fluoro-5-methylchrysene (7-F-5-MeC) (C) after incubation with rat liver 9000 g supernatant. 

Embryos of P. coccineus and P. vulgaris convert Z to (OX)Z and (OX) (diH)Z [24], and both compounds have been shown to occur naturally in the latter. The reduced forms of O Xylosyl derivatives were not detected in P. lunatus embryos. To determine the pathway leading to the reduced conjugate, the metabolites of [ C]-Z and [ C]-(OX)Z were examined after incubation with enzyme extracts obtained from P. coccineus embryos. (OX)Z was not converted to its dihydro derivative whereas Z was converted to (diH)Z under the appropriate reaction conditions (Fig. 3). Therefore, the reduction of the side chain seems to preceed O-xylosylation in P. coccineus embryos. Preliminary experiments to characterize the enzyme converting... 

Additionally, all phase I metabolites were extensively conjugated with D-glucuronic acid and most metabolites detected after longer incubation, were conjugated forms in any test animal species, indicating that phase II reactions also play important roles for metabolism of methoxychlor. Therefore, contribution of phase II metabolism which may (or possibly may not) work as a detoxification process, needs to be taken into account to explain the in vivo toxicity of methoxychlor. 

Figure 15 Use of photodiode array detection of HPLC separation, (a) The photodiode array detector can produce three-dimensional pictures such as that illustrated in this figure, which represents part of an HPLC separation of the metabolites of 25-hydroxydihydrota-chysterolj formed after in vitro incubation with HD-11 cells. The three-dimensional picture suggests that what appears to be a single peak is in fact composed of two umesolved compounds with very different spectra. This can perhaps be more easily appreciated in (b), which shows the HPLC peak monitored at 251 nm and the UV spectra of the leading edge of the peak, the peak apex, and the trailing edge. (From Ref. 7.)...

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