Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine.

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed

The terminal step in vitamin C biosynthesis in Trypanosoma cruzi is mediated by a FMN-dependent galactonolactone oxidase

Humans lack the ability to synthesize vitamin C (ascorbate) due to the absence of gulonolactone oxidase, the last enzyme in the biosynthetic pathway in most other mammals. The corresponding oxidoreductase in trypanosomes therefore represents a target that may be therapeutically exploitable. This is reinforced by our observation that Trypanosoma cruzi, the causative agent of Chagas' disease, lacks the capacity to scavenge ascorbate from its environment and is therefore dependent on biosynthesis to maintain intracellular levels of this vitamin. Here, we show that T. cruzi galactonolactone oxidase (TcGAL) can utilize both L-galactono-γ-lactone and D-arabinono-γ-lactone as substrates for synthesis of vitamin C, in reactions that obey Michaelis–Menten kinetics. It is >20-fold more active than the analogous enzyme from the African trypanosome Trypanosoma brucei. FMN is an essential cofactor for enzyme activity and binds to TcGAL non-covalently. In other flavoproteins, a histidine residue located within the N-terminal flavin-binding motif has been shown to be crucial for cofactor binding. Using site-directed mutagenesis, we show that the corresponding residue in TcGAL (Lys-55) is not essential for this interaction. In contrast, we find that histidine and tryptophan residues (His-447 and Trp-448), localized within a C-terminal motif (HWXK) that is a feature of ascorbate-synthesizing enzymes, are necessary for the FMN association. The conserved lysine residue within this motif (Lys-450) is not required for cofactor binding, but its replacement by glycine renders the protein completely inactive