Selective steroidogenic cytochrome P450 haem iron ligation by steroid-derived isonitriles.

Alkyl isonitriles, R-NC, have previously been shown to ligate the heme (haem) iron of cytochromes P450 in both accessible oxidation states (ferrous, Fe2+, and ferric, Fe3+). Herein, the preparation of four steroid-derived isonitriles and their interactions with several P450s, including the steroidogenic CYP17A1 and CYP106A2, as well as the more promiscuous drug metabolizers CYP3A4 and CYP2D6, is described. It was found that successful ligation of the heme iron by the isonitrile functionality for a given P450 depends on both the position and stereochemistry of the isonitrile on the steroid skeleton. Spectral studies indicate that isonitrile ligation of the ferric heme is stable upon reduction to the ferrous form, with reoxidation resulting in the original complex. A crystallographic structure of CYP17A1 with an isonitrile derived from pregnanalone further confirmed the interaction and identified the absolute stereochemistry of the bound species.

Conformational heterogeneity suggests multiple substrate binding modes in CYP106A2.

CYP106A2 (cytochrome P450meg) is a bacterial enzyme originally isolated from B. megaterium, and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone 15N, 1H and 13C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations.

Standard Adult Gastric Emptying Scintigraphy Criteria Is Applicable for Partial Meal Ingestion.

BACKGROUND/AIMS: Gastric emptying scintigraphy is commonly performed to assess for dysmotility. A standardized meal with associated threshold criteria was established in 2000 to enable robust interpretation. However, no guidance is available to interpret results when patients do not ingest the entire meal. The purpose of this study is to determine the continued appropriateness of the threshold criteria in contemporary clinical practice and its relevance for partially ingested meals. METHODS: This retrospective study analyzed patients (n = 1365 total) who underwent solid-phase gastric emptying scintigraphy at an academic medical center. Patients were stratified based on their completion of the standard meal. Patients were further stratified into normal and delayed gastric emptying cohorts based on the current criteria. Percent gastric retention values at 1, 2, 3, and 4 h were compared. RESULTS: Median (95% upper reference) normal gastric retention values for the complete standard meal were 64% (87%) at 1 h, 25% (60%) at 2 h, 13% (54%) at 3 h and 4% (9%) at 4 h. Consumption of at least 50% of the standard meal yielded similar retention; 53% (86%) at 1 h, 19% (58%) at 2 h, 6% (29%) at 3 h and 3% (10%) at 4 h. There was no significant age- or gender-specific differences using the current criteria, and no differences were observed based on diabetic status. Retention values matched well with the current criteria and validated with data-driven clustering. CONCLUSION: Adult normative standards for gastric emptying scintigraphy are appropriate for differentiating normal and delayed populations and can be applied to partial meals with at least 50% completion.