Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse

Abstract

ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CL(T) (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its V(max⁡)/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3.

Figure 1

Time course of blood ethanol concentration in mice after ethanol administration (i.p.) at various doses. Each plot represents the mean ± SD of 3 mice. ○ 1 g/kg; □ 2 g/kg; ▵ 3 g/kg; ▿ 4.5 g/kg; ⋄ 5 g/kg.

Figure 2

(a) Effect of ethanol dose on elimination rate (β) and normalized AUC (AUC/dose) of blood ethanol. (b) Correlation of normalized AUC with β in mice for various doses of ethanol. β (○) and normalized AUC (□) were calculated from the regression line fitted to the blood ethanol concentrations at each dose in Figure 1.

Figure 3

(a) Effect of ethanol dose on liver ADH activity. Three mice were sacrificed at scheduled times during ethanol metabolism after various doses of ethanol: 0.5, 1, and 2 h for 1 and 2 g/kg (9 mice in each dose); 0.5, 1, 2, 4, and 8 h for 3 g/kg (15 mice in the dose); 0.5, 1. 2, 4, 8, and 12 h for 0, 4.5, and 5.0 g/kg (18 mice in each dose), and livers were then removed to prepare liver extracts. The liver ADH activity was measured by the conventional assay with 15 mM ethanol as a substrate at pH 10.7 using liver extracts and is expressed in terms of liver weight/kg body weight. The activities were averaged in each group of ethanol dose to obtain the mean ± SD. (b) Effect of ethanol dose on ADH 1 (○) and ADH 3 (⚫) content of liver. In addition to liver ADH activity, the liver extracts were used to measure ADH isozyme contents by EIA using isozyme-specific antibodies. Liver ADH isozyme contents were also averaged in each group of ethanol dose to obtain the mean ± SD. (c) Effect of ethanol dose on ratio of ADH 3 content to ADH 1 content.

Figure 4

Correlation of liver ADH activity with ADH 1 (○) and ADH 3 (□) contents of liver. Each plot represents the value obtained from Figures 3(a) and 3(b).

Figure 5

Effect of ethanol dose on catalytic efficiency (V _max⁡/K _m) of liver ADH activity. The apparent V _max⁡ and K _m of liver ADH activity were measured using liver extracts from mice 1 h (○) and 4 h (□) after the administration of each dose of ethanol. V _max⁡ is expressed per mg of the sum of the ADH 1 and ADH 3 contents. Each plot represents the average value of 3 mice.

Figure 6

Correlation of β and body clearance (CL_T) with liver ADH activity. β value (○) was from Figure 2. CL_T value (□) was the reciprocal of the normalized AUC in Figure 2. Liver ADH activity was from Figure 3(a).

Figure 7

Correlation of body clearance (CL_T) with liver ADH 1 and ADH 3 contents. CL_T value was from Figure 6. Liver ADH 1 (○) and ADH 3 (□) contents were from Figure 3(b).

Figure 8

Effect of ethanol dose on theoretical liver ADH 1 and ADH 3 activities in two-ADH-complex model. Liver ADH 1 activity was estimated by multiplying the ADH 1 content by the V _max⁡/mg of ADH 1 (4.0 units/mg). The ADH 3 activity was calculated by subtracting the ADH 1 activity from the total liver ADH activity. The total liver ADH activity was from Figure 3(a) and liver ADH 1 content from Figure 3(b). The theoretical ADH 1 (○) and ADH 3 (□) activities were obtained by normalizing by the ratio of the total ADH activity to that for the control.

Figure 9

Correlation of β and normalized AUC (AUC/dose) of blood ethanol with theoretical ratio of activities of the two ADHs (ADH 3/ADH 1). The values of β (○) and normalized AUC (□) were from Figure 2. Theoretical activities of ADH 1 and ADH 3 were from Figure 8.