Turnover rate of coenzyme A in mouse brain and liver

Abstract

Coenzyme A (CoA) is a fundamental cofactor involved in a number of important biochemical reactions in the cell. Altered CoA metabolism results in severe conditions such as pantothenate kinase-associated neurodegeneration (PKAN) in which a reduction of the activity of pantothenate kinase isoform 2 (PANK2) present in CoA biosynthesis in the brain consequently lowers the level of CoA in this organ. In order to develop a new drug aimed at restoring the sufficient amount of CoA in the brain of PKAN patients, we looked at its turnover. We report here the results of two experiments that enabled us to measure the half-life of pantothenic acid, free CoA (CoASH) and acetylCoA in the brains and livers of male and female C57BL/6N mice, and total CoA in the brains of male mice. We administered (intrastriatally or orally) a single dose of a [13C3-15N-18O]-labelled coenzyme A precursor (fosmetpantotenate or [13C3-15N]-pantothenic acid) to the mice and measured, by liquid chromatography-mass spectrometry, unlabelled- and labelled-coenzyme A species appearance and disappearance over time. We found that the turnover of all metabolites was faster in the liver than in the brain in both genders with no evident gender difference observed. In the oral study, the CoASH half-life was: 69 ± 5 h (male) and 82 ± 6 h (female) in the liver; 136 ± 14 h (male) and 144 ± 12 h (female) in the brain. AcetylCoA half-life was 74 ± 9 h (male) and 71 ± 7 h (female) in the liver; 117 ± 13 h (male) and 158 ± 23 (female) in the brain. These results were in accordance with the corresponding values obtained after intrastriatal infusion of labelled-fosmetpantotenate (CoASH 124 ± 13 h, acetylCoA 117 ± 11 and total CoA 144 ± 17 in male brain).

Fig 1. Levels of free CoA (CoASH), acetylCoA and pantothenic acid (PA) in male mouse A) liver and B) brain after administration of a low PA diet for 3 and 6 days.

*p < 0.02; N.S. No Significance. Error bar, mean ± SD for triplicate samples.

Fig 2

Level of unlabelled pantothenic acid (PA), free CoA (CoASH) and acetylCoA in A) female and B) male mouse liver and C) female and D) male brain after administration of a single oral dose of [¹³C₃-¹⁵N]-PA. C57BL/6N mice were kept for 3 days under a PA-free diet and then orally dosed with 25 mg/kg of [¹³C₃-¹⁵N]-PA. Liver and brain samples were harvested over a collection period of 0–342 h and unlabelled PA, CoASH and acetylCoA level were measured by LC-MS.

Fig 3

Half-life of free CoA (CoASH) and acetylCoA in A) female B) male mouse liver. C57BL/6N mice were kept for 3 days under a PA-free diet and then orally dosed with 25 mg/kg of [¹³C₃-¹⁵N]-PA. Liver samples were collected and analysed by LC-HRMS to determine labelled (+4 AMU) and unlabelled CoASH and acetylCoA levels. The metabolite half-life was calculated from C_max to the last time point collected. Error bars, mean ± SD for triplicate samples.

Fig 4

Half-life of free CoA (CoASH) and acetylCoA in A) female B) male mouse brain. C57BL/6N mice were kept for 3 days under a PA-free diet and then orally dosed with 25 mg/kg of [¹³C₃-¹⁵N]-PA. Brain samples were collected and analysed by LC-HRMS to determine the labelled (+4 AMU) and unlabelled CoASH and acetylCoA levels. The metabolite half-life was calculated from C_max to the last time point collected. Error bars, mean ± SD for triplicate samples.

Fig 5. Formation of labelled-CoA species from labelled-fosmetpantotenate (+6 AMU).

Fig 6

Half-life in male mouse brain of A) free CoA (CoASH) (+4 and+6 AMU), B) acetylCoA (+4 and +6 AMU) and C) total CoA +4 and +6 AMU. C57/Bl6 mice received an intrastriatal injection of 125 μg of +6 AMU labelled-fosmetpantotenate at each brain hemisphere (250 μg total dose) and brain samples were collected over a period of 336 h after dosing. For CoASH +4, acetylCoA +4 and total CoA +4 the time interval 96–336 h was considered for the half-life calculation. For CoASH +6, acetylCoA +6 and total CoA +6 the time interval 24–336 h was used and t = 48 h was excluded from the regression. Error bars, mean ± SD for triplicate samples.