Metabolism of levulinate in perfused rat livers and live rats: conversion to the drug of abuse 4-hydroxypentanoate

Abstract

Calcium levulinate (4-ketopentanoate) is used as an oral and parenteral source of calcium. We hypothesized that levulinate is converted in the liver to 4-hydroxypentanoate, a new drug of abuse, and that this conversion is accelerated by ethanol oxidation. We confirmed these hypotheses in live rats, perfused rat livers, and liver subcellular preparations. Levulinate is reduced to (R)-4-hydroxypentanoate by a cytosolic and a mitochondrial dehydrogenase, which are NADPH- and NADH-dependent, respectively. A mitochondrial dehydrogenase or racemase system also forms (S)-4-hydroxypentanoate. In livers perfused with [(13)C(5)]levulinate, there was substantial CoA trapping in levulinyl-CoA, 4-hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA. This CoA trapping was increased by ethanol, with a 6-fold increase in the concentration of 4-phosphopentanoyl-CoA. Levulinate is catabolized by 3 parallel pathways to propionyl-CoA, acetyl-CoA, and lactate. Most intermediates of the 3 pathways were identified by mass isotopomer analysis and metabolomics. The production of 4-hydroxypentanoate from levulinate and its stimulation by ethanol is a potential public health concern.

FIGURE 1.

Proposed scheme for the metabolism of levulinate and 4-hydroxypentanoate. The hypothetical enzyme activities, designated by numbers in italics are: 1, acid-CoA ligase; 2, hydroxyacid dehydrogenase; 3, 4-hydroxyacyl-CoA dehydrogenase; 4, acyl-CoA dehydrogenase; 5, enoyl-CoA hydratase; 6, 3-hydroxyacyl-CoA dehydrogenase; 7, 3-ketoacyl-CoA thiolase; 8, 4-hydroxyacyl-CoA kinase; 9, acyl-CoA hydrolase; 10, α-oxidation enzymes. The “multiple reactions” mentioned between 4-phosphopentanoyl-CoA and 3-hydroxypentanoyl-CoA result in the isomerization of 4-hydroxypentanoyl-CoA to 3-hydroxypentanoyl-CoA (5).

FIGURE 2.

Profiles of plasma concentrations of levulinate (A) and 4-hydroxypentanoate (B) in rats infused intravenously with sodium levulinate ± ethanol. Data are presented as mean ± S.E. (n = 6).

FIGURE 3.

Effect of ethanol on levulinate uptake and metabolism in perfused rat livers. The lower section of each bar, labeled “To catabolism” was calculated as the difference between the uptake of levulinate and the release of 4-hydroxypentanoate. It corresponds to the disposal of levulinate via Pathways A, B, and B′. Data are presented as mean ± S.E. (n = 6).

FIGURE 4.

Release of [¹³C]formate by rat livers perfused with [¹³C_n]levulinate. Shown are concentrations of [¹³C]formate at the end of 120-min perfusions with [¹³C₅]levulinate (n = 6), [¹³C₅]levulinate + 20 mm ethanol (n = 6), [3-¹³C]levulinate (n = 2), or [1,2,4,5-¹³C₅]levulinate (n = 2).