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. 2015 May;47(5):917-24.
doi: 10.1007/s00726-015-1920-6. Epub 2015 Jan 25.

A (14)C-leucine absorption, distribution, metabolism and excretion (ADME) study in adult Sprague-Dawley rat reveals β-hydroxy-β-methylbutyrate as a metabolite

Anthony J Lee  1 , David W A Beno, Xiaolin Zhang, Robin Shapiro, Mark Mason, Tanita Mason-Bright, Bruce Surber, Neilé K Edens
Affiliations

A (14)C-leucine absorption, distribution, metabolism and excretion (ADME) study in adult Sprague-Dawley rat reveals β-hydroxy-β-methylbutyrate as a metabolite

Anthony J Lee et al. Amino Acids. 2015 May.

Abstract

Leucine is an essential branched-chain amino acid that acts as a substrate for protein synthesis and as a signaling molecule. Leucine not incorporated into muscle protein is ultimately oxidized through intermediates such as β-hydroxy-β-methylbutyrate (HMB) which itself is reported to enhance muscle mass and function in rats and humans. HMB has been reported in the plasma following oral leucine administration in sheep and pigs but not in Sprague-Dawley rats, the standard preclinical model. Therefore, we conducted radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in rats using a low (3 mg/kg) or high dose (1,000 mg/kg) of (14)C-leucine. Blood, tissue, and urine samples were analyzed for (14)C-leucine and its metabolites by HPLC-MS. Our results show for the first time that (14)C-HMB appears in plasma and urine of rats following an oral dose of (14)C-leucine. (14)C-leucine appears in plasma as (14)C-α-ketoisocaproic acid (KIC) with a slower time course than (14)C-HMB, a putative product of KIC. Further, two novel metabolites of leucine were detected in urine, N-acetyl leucine and glycyl leucine. Mass balance studies demonstrate that excretory routes accounted for no more than 0.9 % of the radiolabel and approximately 61 % of the dose was recovered in the carcass. Approximately 65 % of the dose was recovered in total, suggesting that approximately one-third of the leucine dose is oxidized to CO2. In conclusion, this study demonstrates endogenous production of HMB from leucine in adult rats, a standard preclinical model used to guide design of clinical trials in nutrition.

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Figures

Fig. 1
Fig. 1
Metabolism of leucine in mammals. mBCAT mitochondrial branched-chain aminotransferase, BCKDC branched-chain ketoacid dehydrogenase complex, 4-HPPD 4-hydroxy-phenylpyruvatedioxygenase, ECSH1 short-chain enoyl-CoA hydratase. The first intermediate molecule yielded by leucine metabolism, α-ketoisocaproic acid (KIC), is formed when leucine is reversibly transaminated within the muscle by mitochondrial branched-chain amino transferase (mBCAT). Alternatively, a fraction of KIC is decarboxylated and reduced in liver to yield β-hydroxy-β-methylbutyrate (HMB), also known as β-hydroxyisovalerate (CAS 625-08-1). This reaction is catalyzed by ketoisocaproate dioxygenase (4-hydroxyphenylpyruvate dioxygenase; 4-HPPD; EC 1.13.11.27; CAS 9029-72-5) which has been detected in the livers of rat (Sabourin and Bieber 1982) and humans. Under some conditions, HMB can also be formed from leucine by the “ESCH 1 pathway”, which involves dehydrogenation of isovaleryl CoA to methylcrotonyl-CoA, and conversion of methylcrotonyl-CoA to 3-hydroxyisovaleryl-CoA by short-chain enoyl-CoA hydratase (ESCH 1; EC 4.2.1.17)
Fig. 2
Fig. 2
Structure of 2-14C-leucine administered to rats and of 3-14C-HMB used as an HPLC–MS standard. Asterisks denote the location of 14C
Fig. 3
Fig. 3
Distribution of 14C among leucine metabolites in plasma after oral administration of a bolus of 14C-leucine (1,000 mg/kg). Bar height is mean of total radioactivity measured in plasma from two rats. Individual data from each rat are presented numerically on top of each bar. Pooled plasma from each rat pair was fractionated with acetonitrile (ACN) and the percentage of radioactivity in each fraction assessed. The ACN supernatant was analyzed for leucine, KIC, and HMB
Fig. 4
Fig. 4
Representative radiochromatograms showing the metabolite profile in plasma (a, b) and urine (c, d). Values represent concentration after an oral dose of 14C-leucine at 3 mg/kg (a, c) and 1,000 mg/kg (b, d). Plasma values were collected 1 h post dose, while urine values were derived from urine collected for 24 h post dose. Slightly different chromatographic conditions were used for the two doses in plasma; validation experiments showing the recovery of spiked leucine and HMB were performed and confirmed identity of the peaks in the figures (data not shown)
Fig. 5
Fig. 5
Mass-spectrometric identification of leucine metabolites in urine following a single oral administration of 14C-leucine (1,000 mg/kg)
Fig. 6
Fig. 6
Distribution of 14C-leucine among tissues in rats following bolus oral administration of 14C-leucine. Each time point represents the percentage of dose administered to an individual rat. There are two bars per time point to represent the individual rats from each time point
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