Before organogenesis, the avascular embryo is physiologically hypoxic (2-5% O2). Here we hypothesized that, because O2 delivery is limited at this stage of development, excess glucose metabolism could accelerate the rate of O2 consumption, thereby exacerbating the hypoxic state. Because hypoxia can increase mitochondrial superoxide production, excessive hypoxia may contribute to oxidative stress. To test this, we assayed O 2 flux, an indicator of O2 availability, in embryos of glucose-injected hyperglycemic or saline-injected mice. O2 flux was reduced by 30% in embryos of hyperglycemic mice. To test whether hypoxia replicates, and hyperoxia suppresses, the effects of maternal hyperglycemia, pregnant mice were housed in controlled O 2 chambers on embryonic day 7.5. Housing pregnant mice in 12% O 2 , or induction of maternal hyperglycemia (Ͼ250 mg/dl), decreased Pax3 expression fivefold, and increased NTD eightfold. Conversely, housing pregnant diabetic mice in 30% O 2 significantly suppressed the effect of maternal diabetes to increase NTD. These effects of hypoxia appear to be the result of increased production of mitochondrial superoxide, as indicated by assay of lipid peroxidation, reduced glutathione, and H 2O2. Further support of this interpretation was the effect of antioxidants, which blocked the effects of maternal hypoxia, as well as hyperglycemia, on Pax3 expression and NTD. These observations suggest that maternal hyperglycemia depletes O 2 in the embryo and that this contributes to oxidative stress and the adverse effects of maternal hyperglycemia on embryo development.

No abstract

An oxidase-based glucose sensor has been developed that uses a mercaptosilane-modified platinum electrode to achieve selectivity of electrochemical interferants. A platinum-iridium (9:1) wire (0.178 mm o.d., sensing area of 1.12 mm2) is modified with (3-mercaptopropyl)trimethoxysilane. The modified sensors show excellent operational stability for more than 5 days. Glucose oxidase is immobilized on the modified surface (i) by using 3-maleimidopropionic acid as a linker or (ii) by cross-liking with bovine serum albumin using glutaraldehyde. Sensitivities in the range of 9.97 nA/mM glucose are observed when the enzyme is immobilized by method ii. Lower sensitivities (1.13 x 10(-1) nA/mM glucose) are observed when immobilization method i is employed. In terms of linear response range, the sensor enzyme-immobilized by method i is superior to that immobilized by method ii. The linearity is improved upon coating the enzyme layer with polyurethane. The sensor immobilized by method ii and coated with polyurethane exhibits a linear range to 15 mM glucose and excellent selectivity to glucose (0.47 nA/mM) against interferants such as ascorbic acid, uric acid, and acetaminophen.

Extremely acidic aquatic environments,

Glucose gradients generated by an artificial source and -cells were measured using an enzyme-based glucose microsensor, 8-µm tip diameter, as a self-referencing electrode. The technique is based on a difference measurement between two locations in a gradient and thus allows us to obtain real-time flux values with minimal impact of sensor drift or noise. Flux values were derived by incorporation of the measured differential current into Fick's first equation. In an artificial glucose gradient, a flux detection limit of 8.2 ( 0.4 pmol‚cm -2 ‚s -1 (mean ( SEM, n ) 7) with a sensor sensitivity of 7.0 ( 0.4 pA/ mM (mean ( SEM, n ) 16) was demonstrated. Under biological conditions, the glucose sensor showed no oxygen dependence with 5 mM glucose in the bulk medium. The addition of catalase to the bulk medium was shown to ameliorate surface-dependent flux distortion close to specimens, suggesting an underlying local accumulation of hydrogen peroxide. Glucose flux from -cell clusters, measured in the presence of 5 mM glucose, was 61.7 ( 9.5 fmol‚nL -1 ‚s -1 (mean ( SEM, n ) 9) and could be pharmacologically modulated. Glucose consumption in response to FCCP (1 µM) transiently increased, subsequently decreasing to below basal by 93 ( 16 and 56 ( 6%, respectively (mean ( SEM, n ) 5). Consumption was decreased after the application of 10 µM rotenone by 74 ( 5% (mean ( SEM, n ) 4). These results demonstrate that an enzyme-based amperometric microsensor can be applied in the self-referencing mode. Further, in obtaining glucose flux measurements from small clusters of cells, these are the first recordings of the real-time dynamic of glucose movements in a biological microenvironment.