Molecular and Cellular Biochemistry

, Volume 400, Issue 1–2, pp 125–133 | Cite as

Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency

  • Mamoru OhtoyoEmail author
  • Masakazu Tamura
  • Nobuo Machinaga
  • Fumihito Muro
  • Ryuji Hashimoto


Caramel food colorant 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) causes lymphopenia in animals through sphingosine 1-phosphate lyase (SPL) inhibition. However, this mechanism of action is partly still controversial because THI did not inhibit SPL in vitro either in cell-free or in cell-based systems. It is thought that the in vitro experimental conditions which have been used so far were not suitable for the evaluation of SPL inhibition, especially in case of cell-based experiments. We speculated that the key factor might be the coenzyme pyridoxal 5′-phosphate (PLP), an active form of vitamin B6 (VB6), because media used in cell-based assays usually contain an excess amount of VB6 which leads to the activation of SPL. By the use of VB6-deficient culture medium, we could regulate apo- (without PLP) and holo- (with PLP) SPL enzyme in cultured cells, resulting in the successful detection of SPL inhibition by THI. Although the observed inhibitory effect was not as strong as that of 4-deoxypyridoxine (a VB6 analog SPL inhibitor), these findings may be useful for further understanding the mechanism of action of THI.


2-Acetyl-4-(tetrahydroxybutyl)imidazole Sphingosine 1-phosphate S1P lyase Vitamin B6 4-Deoxypyridoxine 



The authors are grateful to Dr. Mika Ikeda for practical advice, project team members for sharing insights, and Human Metabolome Technologies, Inc., for metabolomic analysis. The authors also thank Drs. Ryuta Koishi, Gen Kudo and Hiroshi Yokota for useful suggestions, and Drs. Tohru Takahashi and Hidehiko Furukawa for support.

Supplementary material

11010_2014_2268_MOESM1_ESM.xlsx (80 kb)
Supplementary material 1 (XLSX 79 kb)


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mamoru Ohtoyo
    • 1
    Email author
  • Masakazu Tamura
    • 2
  • Nobuo Machinaga
    • 3
  • Fumihito Muro
    • 4
  • Ryuji Hashimoto
    • 1
  1. 1.New Modality Research Laboratories, R&D DivisionDaiichi Sankyo Co., Ltd.TokyoJapan
  2. 2.Biologics Pharmacology Research Laboratories, R&D DivisionDaiichi Sankyo Co., Ltd.TokyoJapan
  3. 3.Medicinal Chemistry Research Laboratories, R&D DivisionDaiichi Sankyo Co., Ltd.TokyoJapan
  4. 4.Vaccine Business Strategy Department, Vaccine Business Intelligence DivisionDaiichi Sankyo Co., Ltd.TokyoJapan

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