Research Paper Volume 13, Issue 23 pp 24963—24988

Metabolomic profiling of plasma from middle-aged and advanced-age male mice reveals the metabolic abnormalities of carnitine biosynthesis in metallothionein gene knockout mice

Yoshito Kadota1, , Asuka Yano1, , Takashige Kawakami1, , Masao Sato1, , Shinya Suzuki1, ,

  • 1 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan

Received: June 18, 2021       Accepted: November 22, 2021       Published: December 1, 2021
How to Cite

Copyright: © 2021 Kadota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Metallothionein (MT) is a family of low molecular weight, cysteine-rich proteins that regulate zinc homeostasis and have potential protective effects against oxidative stress and toxic metals. MT1 and MT2 gene knockout (MTKO) mice show shorter lifespans than wild-type (WT) mice. In this study, we aimed to investigate how MT gene deficiency accelerates aging. We performed comparative metabolomic analyses of plasma between MTKO and WT male mice at middle age (50-week-old) and advanced age (100-week-old) using liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS). The concentration of N6,N6,N6-trimethyl-L-lysine (TML), which is a metabolic intermediate in carnitine biosynthesis, was consistently higher in the plasma of MTKO mice compared to that of WT mice at middle and advanced age. Quantitative reverse transcription PCR (RT-PCR) analysis revealed remarkably lower mRNA levels of Tmlhe, which encodes TML dioxygenase, in the liver and kidney of male MTKO mice compared to that of WT mice. L-carnitine is essential for β-oxidation of long-chain fatty acids in mitochondria, the activity of which is closely related to aging. Our results suggest that reduced carnitine biosynthesis capacity in MTKO mice compared to WT mice led to metabolic disorders of fatty acids in mitochondria in MTKO mice, which may have caused shortened lifespans.


MT: Metallothionein; ROS: reactive oxygen species; MTKO: MT1 and MT2 knockout; WT: mice wild-type; LC-TOF-MS: liquid chromatography time-of-flight mass spectrometry; BPI: base peak intensity; ESI: electrospray ionization; PCA: principal component analysis; PLS-DA: partial least squares discriminant analysis; OPLS-DA: orthogonal partial least squares-discriminant analysis; VIP: variable importance in projection; HCA: hierarchical clustering analysis; EPA: eicosapentaenoic acid; 12-HETE: 12-hydroxy-eicosatetraenoic-acid; 12(S)-HPETE: 12S-hydroperoxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid; PGE2: prostaglandin E2; 15-keto-13,14-dihydro-PGA2: 15-keto-13,14-dihydro-prostaglandin A2; TXB2: thromboxane B2; 12,13-DHOME: 12,13-dihydroxy-9Z-octadecenoic acid; PPAR: proliferator-activated receptor; HFD: high-fat diet; TCA: tricarboxylic acid; TML: N6,N6,N6-trimethyl-L-lysine; CPT: carnitine palmityltransferase; XOR: xanthine oxidoreductase; γ-BB: γ-butryobetaine; 13(S)-HPODE: 13-L-hydroperoxylinoleic acid; EPEA: eicosapentaenoyl ethanolamide; PKU: phenylketonuria; PAH: phenylalanine hydroxylase; TMLD: TML dioxygenase; HTML: 3-hydroxy-TML; Tmlhe: TML hydroxylase, epsilon; HTMLA: HTML aldolase; TMABA: 4-N-trimethylaminobutyraldehyde; THA1: threonine aldolase 1; SHMT: serine hydroxymethyltransferase; TMABADH: TMABA dehydrogenase; Aldh9a1: aldehyde dehydrogenase 9 subfamily A1; BBD: γ-BB dioxygenase; Bbox1: 2-oxoglutarate dioxygenase 1; DENA: diethylnitrosamine; RT: reverse transcription.