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Plasma levels of 1,5-anhydroglucitol (1-deoxyglucose), a short-term marker of glycemic control, have been measured and used clinically in Japan since the early 1990s. Plasma levels of 1,5-anhydroglucitol are typically measured using either a commercially available enzymatic kit or GC/MS. A more sensitive method is needed for the analysis of 1,5-anhydroglucitol in urine, where levels are significantly lower than in plasma. We have developed a sensitive and selective LC/MS(3) assay utilizing hydrophilic interaction liquid chromatography and ion trap mass spectrometry for the quantitative determination of 1,5-anhydroglucitol in human urine. Diluted human urine samples were analyzed by LC/MS(3) using an APCI source operated in the negative ionization mode. Use of an ion trap allowed monitoring of MS(3) transitions for both 1,5-anhydroglucitol and the internal standard which provided sufficient selectivity and sensitivity for analysis from 50 microL of human urine. Quantitation of 1,5-anhydroglucitol levels in urine was accomplished using a calibration curve generated in water (calibration range 50 ng/mL to 10 microg/mL). Method ruggedness and reproducibility were evaluated by determining the intra- and inter-day accuracies and precision of the assay, as well as the bench-top and freeze-thaw stability. For both inter- and intra-assay evaluations, the accuracy of the assay was found to be acceptable, with the concentrations of all QCs tested not deviating more than 8% from theoretical. Four-hour bench-top and freeze-thaw stabilities were also evaluated; 1,5-anhydroglucitol was found to be stable at room temperature (<18% deviation from theoretical) and during 3 freeze-thaw cycles (<1% deviation from theoretical, except at the lowest QC level). The LC/MS(3) assay was then used to successfully determine the concentration of 1,5-AG in more than 200 urine samples from diabetic patients enrolled in a clinical study.

Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo. The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.