Enzymatic determination of b-galactose by b-galactose dehydrogenase has been a useful method to determine b-galactose in biological substances [1,2] since the enzyme was identified and purified from Pseudomonas saccharophila by Doudoroff et al. [3] in the 1950s. This method was based on the b-galactose dehydrogenase catalysis of the oxidation of galactose in the presence of oxidized b-nicotinamide adenine dinucleotide (NAD) to produce galactonolactone and the reduced form of bnicotinamide adenine dinucleotide (NADH) which can be detected at 340 nm. The production of NADH is proportional to the concentration of galactose [1–3]. In this study, we optimized the enzymatic method for galactose determination and investigated galactose metabolism in liver spheroids, HepG2 spheroids, and HepG2 cells cultured in monolayer. To determine an optimal pH range, the conditions of 1mg/ml b-galactose, 4U/ml b-galactose dehydrogenase, and 16mM b-nicotinamide adenine dinucleotide in the pH 5, 6, 7, 8, 9, and 10 buffers (Na2HPO4=KH2PO4 buffer, 200mM) were tested. The assays including all following assays were performed in a 96-well format and absorbance was read on a microplate reader (Multiscan RC, Labsystems). After 50min incubation at 37 C, the reactions in pH 7–10 buffers (Na2HPO4=KH2PO4 buffer, 200mM) were completed, whereas the reactions in these buffers at pH 5 and 6 slowly increased. The best results were obtained between pH 8 and 9. This indicates that b-galactose dehydrogenase works well in an alkaline condition. pH 8 buffer was used in the following assays in this study. At the concentrations of 1 and 2U/ml b-galactose dehydrogenase in the conditions of 1mg/ml galactose and 16mM NAD, the reactions were completed at 70 and 90min, respectively. The maximum absorbance of both concentrations was 20–25% lower than that in 4U/ml b-galactose dehydrogenase. A suitable ratio of substrate concentration and enzyme concentration is essential for an optimal enzymatic reaction. Based on this result, 4U/ml b-galactose dehydrogenase was utilized for all the following experiments. Fig. 1 shows the effect of NAD concentration on catalysis of b-galactose dehydrogenase. As shown in Fig. 1, the concentration of NAD modified the enzymatic reaction rate. A higher concentration of NAD produced a higher OD value after the completion of the reaction although all three concentrations of NAD tested were high enough to receive hydrogen from 1mg/ml galactose in the defined method. Therefore, a concentration of NAD between 12 and 16mM is recommended. The concentration of 16mM was used for the following experiments. The catalytic kinetics and changes of OD readings are shown in Fig. 2A. The lower the substrate concentration, the sooner the reaction was completed as indicated by OD values. At 50min and the concentration of 1mg/ ml galactose or lower, the absorbance no longer increased with time, indicating completion of the reaction. In the higher concentrations of galactose, the absorbance slowly increased until 110min. Once the absorbance reached the peak value, it decreased slowly and steadily. The average decrease of OD values was only 0.44% every 10min and a total 3.54% decrease was detected 80min after peak time point. This indicates that the end product, NADH, is stable enough to allow a flexible assay. The linearity of galactose concentrations versus OD values after 60min reaction is shown in Fig. 2B. When the OD value was lower than 1.8 (equivalent to 1mg/ml galactose or 5.5mM NADH), the OD values were linear Analytical Biochemistry 311 (2002) 179–181