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Protein-bound uremic toxins in hemodialysis patients measured by liquid chromatography/tandem mass spectrometry and their effects on endothelial ROS production

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Abstract

Cardiovascular disease (CVD) is prevalent in patients with chronic kidney disease (CKD). In hemodialysis (HD) patients, some protein-bound uremic toxins are considered to be associated with CVD. However, it is not yet known which uremic toxins are important in terms of endothelial toxicity. Serum samples were obtained from 45 HD patients before and after HD. Total and free serum concentrations of indoxyl sulfate, indoxyl glucuronide, indoleacetic acid, p-cresyl sulfate, p-cresyl glucuronide, phenyl sulfate, phenyl glucuronide, phenylacetic acid, phenylacetyl glutamine, hippuric acid, 4-ethylphenyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) were simultaneously measured by liquid chromatography/electrospray ionization–mass spectrometry/mass spectrometry (LC/ESI-MS/MS). The effects of these solutes at their pre-HD mean and maximum serum concentrations on reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVEC) were measured with a ROS probe. Serum levels of 11 of the solutes (all except 4-ethylphenyl sulfate) were significantly increased in HD patients compared to healthy subjects. All 12 solutes showed changes in their protein-binding ratios. In particular, indoxyl sulfate, p-cresyl sulfate, CMPF, and 4-ethylphenyl sulfate showed high protein-binding ratios (>95 %) and low reduction rates by HD (<35 %). Indoxyl sulfate at its mean and maximum pre-HD serum concentrations—even with 4 % albumin—stimulated ROS production in HUVEC most intensely, followed by CMPF. In conclusion, the serum levels of 11 protein-bound uremic toxins were increased in HD patients. Indoxyl sulfate, p-cresyl sulfate, and CMPF could not be removed efficiently by HD due to their high protein-binding ratios. Indoxyl sulfate most intensely induced endothelial ROS production, followed by CMPF.

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Conflict of interest

Y.I., A.E., and K.K. are employed by Kureha Corporation. The other authors declare no competing interests.

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Authors and Affiliations

  1. Biomedical Research Laboratories, Kureha Corporation, 3-2-2, Nihonbashi-Hamacho, Chuo-ku, Tokyo, 103-8552, Japan

    Yoshiharu Itoh, Atsuko Ezawa & Kaori Kikuchi

  2. Meiyo Clinic, Hemodialysis Center, 64-3. Yadouri-cho, Toyohashi, Aichi, 441-8023, Japan

    Yoshinari Tsuruta

  3. Department of Advanced Medicine for Uremia, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Toshimitsu Niwa

Authors
  1. Yoshiharu Itoh
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  2. Atsuko Ezawa
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  3. Kaori Kikuchi
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  4. Yoshinari Tsuruta
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  5. Toshimitsu Niwa
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Correspondence to Toshimitsu Niwa.

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Published in the special paper collection Biomedical Mass Spectrometry with guest editors Toyofumi Nakanishi and Mitsutoshi Setou.

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Itoh, Y., Ezawa, A., Kikuchi, K. et al. Protein-bound uremic toxins in hemodialysis patients measured by liquid chromatography/tandem mass spectrometry and their effects on endothelial ROS production. Anal Bioanal Chem 403, 1841–1850 (2012). https://doi.org/10.1007/s00216-012-5929-3

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  • DOI: https://doi.org/10.1007/s00216-012-5929-3

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