Optimization of a Single Substrate-Based Fluorometric Assay for Glucose and Lactate Measurement to Assess...

A fluorometric assay for single substrate-based glucose and lactate measurements was demonstrated by adopting an already established protocol for glucose and by optimizing pH, enzyme concentration, substrate concentration, and types of buffers for lactate. Linear calibration curves for glucose and lactate concentrations from 1 to 100 μM were obtained with correlation coefficients (R²) of 0.94 and 0.98, respectively. First, with the optimized protocol, single embryo quality was successfully evaluated. Three different initial stages of embryos (n = 58) were cultured for 24 h, and glycolytic activities were calculated by measuring amounts of glucose consumption and lactate production. Results showed that embryos cultured at a later stage had lower glycolytic activities, implying more developmental activities. Second, glucose and lactate concentrations in blood plasma of diet-induced obese (DIO) mice were measured. Levels of both glucose and lactate in DIO mice were higher than those in normal mice by 2.15 and 3.8 mmol/L, respectively (both p < 0.001). Finally, clinical serum samples were analyzed and categorized into three groups based on their measured glucose concentrations: normal (4.73 ± 0.29 mmol/L), prediabetic (6.49 ± 0.13 mmol/L), and diabetic (11.34 ± 1.36 mmol/L) (p < 0.05). Collectively, this developed technique can be used to select a high-quality embryo for transfer as well as to measure glucose and lactate levels in other biological samples.