Ethyl glucuronide (ethyl beta-D-6-glucosiduronic acid), a minor ethanol metabolite in serum or urine, was determined by gas chromatography-mass spectrometry. Prior to this, ethyl glucuronide was synthesized by the reaction of acetobromo-glucosiduronic acid with ethanol. For the determination of ethyl glucuronide, serum samples were precipitated with acetone, and urine specimens were analyzed after evaporation to dryness. The residues were derivatized with acetic anhydride. Capillary gas chromatography was used to find a retention index value of 1920 for the triacetyl derivative. The mass spectrum of the acetylated ethyl glucuronide was recorded. The calibration is linear in the range investigated (0.1-150 mg/L), and the detection limit is 0.1 mg/L. In individual specimens containing between 0.1 and 4 g ethanol per liter serum, ethyl glucuronide could be detected at concentrations between 3 and 14 mg/L and in the corresponding urine specimens at concentrations between 3 and 130 mg/L.

The biochemical properties of a new tungsten-containing aldehyde oxidoreductase from the mesophilic betaproteobacterium Aromatoleum aromaticum EbN1 (AORAa) are presented in this study. The enzyme was purified from phenylalanine-grown cells of an overexpressing mutant lacking the gene for an aldehyde dehydrogenase normally involved in anaerobic phenylalanine degradation. AORAa catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD+ as electron acceptors. In contrast to previously known AORs, AORAa is a heterohexameric protein consisting of three different subunits, a large subunit containing the W-cofactor and an Fe-S cluster, a small subunit containing four Fe-S clusters, and a medium subunit containing an FAD cofactor. The presence of the expected cofactors have been confirmed by elemental analysis and spectrophotometric methods. AORAa has a pH optimum of 8.0, a temperature optimum of 40°C and is completely inactive at 50°C. Compared to archaeal AORs, AORAa is remarkably resistant against exposure to air, exhibiting a half-life time of 1 h as purified enzyme and being completely unaffected in cell extracts. Kinetic parameters of AORAa have been obtained for the oxidation of one aliphatic and two aromatic aldehydes, resulting in about twofold higher kcat values with benzyl viologen than with NAD+ as electron acceptor. Finally, we obtained evidence that AORAa is also catalyzing the reverse reaction, reduction of benzoate to benzaldehyde, albeit at very low rates and under conditions strongly favoring acid reduction, e.g., low pH and using Ti(III) citrate as electron donor of very low redox potential. AORAa appears to be a prototype of a new subfamily of bacterial AOR-like tungsten-enzymes, which differ from the previously known archaeal AORs mostly by their multi-subunit composition, their low sensitivity against oxygen, and the ability to use NAD+ as electron acceptor.

The kinetic profile of ethanol and ethyl glucuronide (EtG) in serum was investigated in three subject groups: 1) Healthy, moderately drinking volunteers (daily intake less than 30 g ethanol) who ingested a single dose of ethanol. In this group the maximum of serum ethyl glucuronide concentration (SEtGC) and of serum ethanol concentration (SEC) did not exceed 3.7 mg/L and 1.5 g/L respectively. EtG peaked 2 to 3.5 h later than ethanol. EtG was eliminated with a terminal half-life of 2 to 3 h. EtG decreased slower than ethanol—the metabolite could still be determined in serum up to 8 h after complete ethanol elimination. 2) In serum samples of teetotalers neither ethanol nor EtG could be found. 3) In 37 of 50 serum samples of drivers suspected of driving under the influence of ethanol, SEtGC was found between the limit of detection (0.1 mg/L) and 20 mg/L. If the SEC is less than 1 g/L and the SEtGC is significantly higher than 5 mg/L, we assume alcohol misuse.

Natural rubber [poly-(cis-1,4-isoprene)] is cleaved to by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA has two c-type haem centres that show two distinct a-bands at 549 and 553 nm in the dithionite-reduced state. A wellresolved midpoint potential (E 0 9) of -65 mV was determined for one haem by spectrophotometric titrations in the absence of dioxygen with dithionite and ferricyanide as reductant and oxidant, respectively. The midpoint potential of the second haem was not resolvable (E 0 9 about "130 to -160 mV). One of the two haems was reduced by NADH (549 nm a-band), similar to bacterial dihaem peroxidases. Evidence for an electron transfer between the two haems was provided by slow reduction of the second haem (553 nm a-band) upon incubation of the partially reduced enzyme at room temperature. Addition of imidazole or related compounds to RoxA led to UV/vis spectral features similar to those observed for partially reduced RoxA. Notably, reduction of RoxA with dithionite or NADH, or binding of compounds such as imidazole, resulted in a reversible inactivation of the enzyme, unlike dihaem peroxidases. In line with this result, RoxA did not show any peroxidase activity. EPR spectra of RoxA as isolated showed two low-spin Fe(III) haem centres, with apparent g-values of 3.39, 3.09, 2.23, 1.92 and 1.50. A weak signal in the g56 region resulting from a high-spin Fe(III) haem was also observed with a preparation-dependent intensity that disappeared in the presence of imidazole. Attempts to provide spectroscopic evidence for binding of the natural substrate (polyisoprene latex) to RoxA failed. However, experimental data are presented that RoxA is able to subtract redox equivalents from its substrate or from model compounds. In conclusion, RoxA is a novel type of dihaem dioxygenase with features clearly different from classical cytochrome c peroxidases.