α-Keto acid metabolism by hepatocytes cultured in a hybrid liver support bioreactor

Abstract

Isolated pig liver cells cultured using a perfusion technique were analyzed over 39 days to test their ability to change the perfusate alpha-keto acid profile. While the pyruvate concentration in the culture medium decreased as of the first day, the alpha-ketoglutarate (KG), alpha-ketoisocaproate (KIC), alpha-ketoisovalerate (KIV) and alpha-keto-beta-methyl-n-valerate (KMV) were synthesized immediately and released by the liver cells. The metabolic capacity of the cell culture system increased up to day 10, decreased during the following 5 days and reached a steady state beyond day 15, which was maintained for at least 30 days. The branched chain alpha-keto acid release, in particular alpha-ketoisocaproate, reflects an effective transamination capacity of the newly developed culture system and shows an intact protein biosynthesis for at least 30 days in vitro.

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

“…Since intracellular pyruvate increases the concentrations of α-ketoglutarate as well as glutamine, glutamate, asparagine, aspartate, alanine, arginine, ornithine, glycine and serine were also significantly elevated in a dose as well as duration of exposure dependent manner. A more precise look at the constitution of PMN amino and α-keto acid changes favour the hypothesis that increases in neutrophil free pyruvate concentrations are mainly followed by pyruvate conversion into important amino and α-keto acid derivatives (Curi et al 1989, 1988; Fuchs et al 1994; Mühling et al 2010, 2007, 2005, 2002). Indeed, our assumption that this metabolisation processes also occurs within PMN cells, may particularly be supported with regard to very similar immunonutritional findings induced by α-ketoglutarate, alanyl-glutamine, ornithine or arginine in other leukocyte cells or even neutrophils.…”

“…For each sample, 5 mg of OPD was dissolved in 5 ml of 3 M HCl (σ) and 10 μl of 2-mercaptoethanol (σ) was added to yield OPD-HCl-ME. This reagent solution was stable for several hours without loss in sensitivity (Fuchs et al 1994). …”

“…via glycolysis in which one molecule of glucose breaks down into two molecules of pyruvate or reversible transamination reactions, etc. ), but also unites several key metabolic processes (Agam and Gutman 1972; Curi et al 1989, 1988; Fauth et al 1993 and 1990; Frei et al 1975; Fuchs et al 1994; Homem de Bittencourt et al 1993; Ing et al 1997; Newsholme et al 1987; Wu et al 2005 and 2003). …”

“…As quoted above, another important step in the formation or use of pyruvate is catalysed by rapid and reversible transamination reactions, for example catalysed via alanine transaminase, producing α-ketoglutarate and alanine (Cicalese et al 1996a, 1996b; Edlund and Halestrap 1988; Gardner and Leese 1988; Selvaraj and Sbarra 1966; Wiemer et al 1995). For leukocytes this is of major importance, because α-ketoglutarate, converted from pyruvate in particular, is responsible for key metabolic and synthetic roles which are essential in cellular viability (Curi et al 1988; Gabbianelli et al 2007; Mahiout et al 1997; Miwa et al 2000; Ramakrishnan et al 1998; Zhou and Yu 1998) and can be very rapidly into a precursor of numerous amino acids, synthetic metabolic intermediates as well as macromolecular synthesis (Curi et al 1989, 1988; Fuchs et al 1994; Riedel et al 1992; Serrano and Curi 1989; Venizelos and Hagenfeldt 1985). Interesting findings suggest indeed that α-ketoglutarate, which is also one of the most important intracellular nitrogen transporters, is not only a key intermediate in the TCA cycle (coming after isocitrate and before succinyl CoA) but also can replenish the cycle in anaplerotic reactions.…”

1H NMR spectroscopy as a tool to evaluate key metabolic functions of primary porcine hepatocytes after cryopreservation

“…The central player in transamination reactions is of course glutamate, which accepts the amine radical of the a-ketoacids during their catabolism. 35 Net production of glutamate was observed in all culture supernatants in CP and FH hepatocytes. Glutamine is produced from glutamate when there is a need for scavanging of ammonium cations.…”