Metabolism of Levulinate in Perfused Rat Livers and Live Rats

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-phosphopentanoylCoA. 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.Levulinate (4-ketopentanoate) is a food additive allowed by the Food and Drug Administration. As a calcium salt, it has been used for many years as an oral and intravenous form of calcium administration (1, 2). It is listed in the pharmacopeias of a number of countries (USA, Europe, India, China, Japan, etc). It is also listed in quality control documents of the World Health Organization. The dry salt is sold on the internet as a non-prescription dietary supplement. To the best of our review of the literature, the catabolism of levulinate has not been investigated in mammalian cells. We found one report on the identification of the reduced form of levulinate, 4-hydroxypentanoate, in the urine of children with ␤-ketothiolase deficiency treated with intravenous calcium levulinate (3). We also found an unexplained report of induction of lactic acidosis in premature babies treated with oral calcium levulinate for hypocalcemia (4).Our interest in levulinate arose from our recent study of the metabolism of 4-hydroxyacids, which are products of lipid peroxidation (C 9 , C 6 ) or drugs of abuse (C 4 , C 5 ) (5, 6). We showed that 4-hydroxyacids with 5 or more carbons are metabolized by two new pathways. The first pathway involves the isomerization of 4-hydroxyacyl-CoAs to 3-hydroxyacylCoAs via 4-phosphoacyl-CoAs, a new class of acyl-CoA esters. After isomerization, the 3-hydroxyacyl-CoAs are catabolized via classical ␤-oxidation to acetyl-CoA and, in the case of odd-chain 4-hydroxyacids, to propionyl-CoA. The second pathway involves a sequence of ␤-, ␣-, and ␤-oxidation steps leading to acetyl-CoA, formate and, in the case of even chain 4-hydroxyacids, to propionyl-CoA.The five-carbon 4-hydroxypentanoate, as a racemic mixture, is a new drug of abuse used in lieu of 4-hydroxybutyrate, which is now a controlled substance in the United States (7-11). Based on our previous work...

DNA Damage Response and Autophagy: A Meaningful Partnership

“…Thus, basal autophagy inhibits IL-1β secretion by degrading both inflammasome proteins (Shi et al, 2012) and pro-IL-1β (Harris et al, 2011). In contrast, under conditions of dual inflammasome and autophagy activation, IL-1β secretion increases through a non-canonical secretory pathway that depends on autophagy components (Dupont et al, 2011).…”

Autophagy and Mechanisms of Effective Immunity

“…This is particularly well described for interleukin (IL)-1β and IL-18, where the absence of autophagy exacerbates their secretion by murine macrophages when stimulated with pro-inflammatory compounds. Specifically, this has been described for beclin1 +/− (Nakahira et al, 2011), LC3b −/− (Nakahira et al, 2011), Atg16L1 −/− (Saitoh et al, 2008), and Atg7 −/− (Saitoh et al, 2008) macrophages or following expression of inactive Atg4B or due to treatment with the chemical autophagy inhibitor 3-methyladenine (3-MA; Saitoh et al, 2008; Harris et al, 2011). Enhanced secretion of IL-1β also occurs when human peripheral blood mononuclear cells are stimulated in the presence of an impaired autophagy pathway due to treatment with 3-MA (Crisan et al, 2011).…”

“…Enhanced secretion of IL-1β also occurs when human peripheral blood mononuclear cells are stimulated in the presence of an impaired autophagy pathway due to treatment with 3-MA (Crisan et al, 2011). Conversely, stimulation of murine DC with rapamycin suppresses IL-1β production in response to inflammatory stimuli (Harris et al, 2011). Rapamycin administration in vivo , inhibits the detected increase in IL-1β but not IL-12, in the serum 4 h following lipopolysaccharide (LPS) treatment (Harris et al, 2011).…”

“…Conversely, stimulation of murine DC with rapamycin suppresses IL-1β production in response to inflammatory stimuli (Harris et al, 2011). Rapamycin administration in vivo , inhibits the detected increase in IL-1β but not IL-12, in the serum 4 h following lipopolysaccharide (LPS) treatment (Harris et al, 2011). Not all stimuli provoke excessive IL-1β secretion in the absence of autophagy.…”

“…Following inflammasome induction, components of the absent in melanoma 2 (AIM2) inflammasome associate with p62 and beclin1 and exhibit K63-linked ubiquitination (Shi et al, 2012). Other examples of the direct targeting of inflammatory proteins by autophagy include the co-localization of the autophagosomal protein LC3 and IL-1β in punctate autophagosomal structures following stimulation of bone marrow-derived macrophages with a variety of TLR ligands including LPS, Pam3Cys, poly (I:C), R837, or CpG (Harris et al, 2011). The authors suggest that autophagy limits IL-1β secretion by facilitating its degradation in the auto-lysosome.…”