Assay of Protein-Bound Lipoic Acid in Tissues by a New Enzymatic Method

doi:10.1006/abio.1998.2615

Analytical Biochemistry

Volume 258, Issue 2, 1 May 1998, Pages 299-304

https://doi.org/10.1006/abio.1998.2615 Get rights and content

Abstract

A new enzymatic method for the determination of protein-bound lipoic acid was established. Bound lipoyl groups were liberated in the form of lipoyllysine by protease digestion and assayed by lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.8.1.4)-mediated NADH oxidation. NADH oxidation was coupled to reduction of the lipoyl disulfide group. Fluorescence kinetics of NADH oxidation were markedly enhanced by the addition of glutathione disulfide, recycling the enzyme-mediated lipoyl/dihydrolipoyl conversion. In the presence of a large excess of glutathione disulfide, NADH oxidation follows pseudo-first-order kinetics in terms of lipoyllysine concentration. A good linear correlation is obtained between the oxidation rate and lipoyllysine concentration up to 5 μM and the calibration curve indicates that the detection limit could be 100 nM lipoyllysine. The method was applied to protease lysates of bovine, rat, and rabbit tissues to determine lipoyllysine levels. Kidney and liver were found to have the highest content of lipoyllysine in the range of 3.9 to 4.6 nmol/g rat or rabbit wet tissue or 11.6 to 13.1 nmol/g bovine acetone powder.

References (32)

J.M. Jilka et al.
J. Biol. Chem.
(1986)
L. Packer et al.
Free Radical Biol. Med.
(1995)
R. Sumathi et al.
Pharmacol. Res.
(1993)
A. Constantinescu et al.
J. Biol. Chem.
(1993)
S. Matsugo et al.
Biochem. Biophys. Res. Commun.
(1995)
S. Matsugo et al.
Biochem. Biophys. Res. Commun.
(1996)
V.E. Kagan et al.
Biochem. Pharmacol.
(1992)
A. Bast et al.
Biochim. Biophys. Acta
(1988)
R.M. Lozano et al.
Arch. Biochem. Biophys.
(1994)
K. Yoshikawa et al.
J. Chromatogr.
(1996)

H. Kataoka et al.

J. Chromatogr.

(1993)

R.H. White

Anal. Biochem.

(1981)

J.C. Shih et al.

Anal. Biochem.

(1981)

F. Tietze

Anal. Biochem.

(1969)

J. Teichert et al.

J. Chromatogr.

(1995)

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Citation Excerpt :
LA is found in various dietary sources at the low µg/g range. Animal tissues with the highest lipoate levels include kidney, heart and liver [7], while it also occurs in vegetables at comparable amounts [8]. The highest lipoyllysine content was detected in spinach, followed by broccoli and tomato [9].
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Regular ArticleAssay of Protein-Bound Lipoic Acid in Tissues by a New Enzymatic Method

Abstract

J. Biol. Chem.

Free Radical Biol. Med.

Pharmacol. Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

J. Chromatogr.

J. Chromatogr.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr.

Regular Article
Assay of Protein-Bound Lipoic Acid in Tissues by a New Enzymatic Method