Abnormal homocysteine metabolism and glutathione depletion in yolk sacs and embryos of diabetic rats


      Exposure to maternal hyperglycemia induces both an increase in oxidative stress and a decrease in glutathione (GSH) in the developing embryo. We hypothesized that the resulting pro-oxidant microenvironment during vulnerable stages of organogenesis may contribute to hyperglycemia-induced malformations.

      Study design

      Diabetes was induced in 8-week-old female rats by IV streptozotocin injection. Glucose was monitored until diabetic levels of hyperglycemia (>250 mg/dL) were achieved. A sustained-release insulin pellet was then inserted subcutaneously. Rats were mated after normal and stable glucose levels (80-150 mg/dL) had been attained. On gestational day (GD) 4, the implants were withdrawn. Experimental rats were sacrificed on GD12 and embryos and yolk sacs were examined morphologically and stored at −80°C until analysis. Intracellular levels of oxidized (GSSG) and reduced glutathione (GSH) and the metabolic precursors, homocysteine (Hcy), methionine, cysteine (reduced), and cystine (oxidized) were analyzed by HPLC with electrochemical detection.


      A 2-fold decrease in the redox ratio (GSH/GSSG) was observed in the malformed compared to normal embryos. In the yolk sac, a 3-fold decrease in GSH/GSSG ratio was observed in malformed embryos, indicating a significant increase in intracellular oxidative stress. A decrease in the glutathione precursors, methionine and cysteine, was associated with decreased GSH levels. The increase in Hcy and cysteine is consistent with hyperglycemia-induced inhibition of γ-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis.


      These data provide strong evidence that glutathione-mediated antioxidant defense mechanisms are compromised in embryos and yolk sacs subjected to maternal hyperglycemia during critical stages of organogenesis. The resulting pro-oxidant intracellular environment is considered critical in the genesis of diabetic embryopathy.