Administration of N-acetylcysteine causes beneficial posttranslational modifications of transthyretin in hemodialysis patients

Publikation: Konferencebidrag uden forlag/tidsskriftPosterForskning

Resumé

INTRODUCTION AND AIMS:
The thiol antioxidant N-acetylcysteine (NAC) may mediate interactions with protein-associated cysteine residues, however, information on protein level in vivo are missing. Therefore, in the present study we aimed to analyze N-acetylcysteine-induced modifications of the protein transthyretin (TTR) in plasma from hemodialysis patients in a randomized, placebo-controlled study in vivo and after administration to plasma in vitro. TTR was selected due to its low molecular weight and the free cysteine residue in the polypeptide chain, which is known to be extensively modified by formation of mixed disulfides.

METHODS:
Plasma levels of TTR were determined by a non-commercial enzyme-linked immunosorbent assay (ELISA) using polyclonal rabbit anti-human TTR antibodies. Spectra of immunoprecipitated TTR were obtained using an AutoflexSpeed matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker-Daltonik, Bremen, Germany). The samples were analyzed in triplicates. For ionization, a Smartbeam-II laser was used and 1500 shots per spot were collected. For spectra calibration an external standard was used. Spectra were evaluated using the software flexAnalysis. TTR variants were expressed as relative amounts of the summed intensity of all observed TTR variants.

RESULTS:
The administration of NAC during a hemodialysis session resulted in a substantial increase of native TTR from median 15% (range 8.8-30%) to median 40% (37-50) and a reduction of S-cysteinylated TTR [51% (44-60) vs. 6.6% (2.4-10)]. Additionally the pronounced formation of a TTR-NAC adduct was detected. However, all these modifications seemed to be reversible. Additionally, in vitro incubation of plasma with NAC confirmed the in vivo results and indicated that changes in PTM pattern of TTR were a function of NAC concentration.

CONCLUSIONS:
We conclude that the interaction of N-acetylcysteine with proteins may explain altered protein functions due to beneficial modification of cysteine residues in hemodialysis patients.
OriginalsprogEngelsk
Publikationsdato2013
StatusUdgivet - 2013

Fingeraftryk

Prealbumin
Acetylcysteine
Proteins
Germany
Molecular Weight
Placebos
Rabbits

Citer dette

@conference{36c3196042ea4c43b5be498c4fcea04a,
title = "Administration of N-acetylcysteine causes beneficial posttranslational modifications of transthyretin in hemodialysis patients",
abstract = "INTRODUCTION AND AIMS:The thiol antioxidant N-acetylcysteine (NAC) may mediate interactions with protein-associated cysteine residues, however, information on protein level in vivo are missing. Therefore, in the present study we aimed to analyze N-acetylcysteine-induced modifications of the protein transthyretin (TTR) in plasma from hemodialysis patients in a randomized, placebo-controlled study in vivo and after administration to plasma in vitro. TTR was selected due to its low molecular weight and the free cysteine residue in the polypeptide chain, which is known to be extensively modified by formation of mixed disulfides.METHODS:Plasma levels of TTR were determined by a non-commercial enzyme-linked immunosorbent assay (ELISA) using polyclonal rabbit anti-human TTR antibodies. Spectra of immunoprecipitated TTR were obtained using an AutoflexSpeed matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker-Daltonik, Bremen, Germany). The samples were analyzed in triplicates. For ionization, a Smartbeam-II laser was used and 1500 shots per spot were collected. For spectra calibration an external standard was used. Spectra were evaluated using the software flexAnalysis. TTR variants were expressed as relative amounts of the summed intensity of all observed TTR variants.RESULTS:The administration of NAC during a hemodialysis session resulted in a substantial increase of native TTR from median 15{\%} (range 8.8-30{\%}) to median 40{\%} (37-50) and a reduction of S-cysteinylated TTR [51{\%} (44-60) vs. 6.6{\%} (2.4-10)]. Additionally the pronounced formation of a TTR-NAC adduct was detected. However, all these modifications seemed to be reversible. Additionally, in vitro incubation of plasma with NAC confirmed the in vivo results and indicated that changes in PTM pattern of TTR were a function of NAC concentration.CONCLUSIONS:We conclude that the interaction of N-acetylcysteine with proteins may explain altered protein functions due to beneficial modification of cysteine residues in hemodialysis patients.",
author = "Alexandra Scholze",
year = "2013",
language = "English",

}

TY - CONF

T1 - Administration of N-acetylcysteine causes beneficial posttranslational modifications of transthyretin in hemodialysis patients

AU - Scholze, Alexandra

PY - 2013

Y1 - 2013

N2 - INTRODUCTION AND AIMS:The thiol antioxidant N-acetylcysteine (NAC) may mediate interactions with protein-associated cysteine residues, however, information on protein level in vivo are missing. Therefore, in the present study we aimed to analyze N-acetylcysteine-induced modifications of the protein transthyretin (TTR) in plasma from hemodialysis patients in a randomized, placebo-controlled study in vivo and after administration to plasma in vitro. TTR was selected due to its low molecular weight and the free cysteine residue in the polypeptide chain, which is known to be extensively modified by formation of mixed disulfides.METHODS:Plasma levels of TTR were determined by a non-commercial enzyme-linked immunosorbent assay (ELISA) using polyclonal rabbit anti-human TTR antibodies. Spectra of immunoprecipitated TTR were obtained using an AutoflexSpeed matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker-Daltonik, Bremen, Germany). The samples were analyzed in triplicates. For ionization, a Smartbeam-II laser was used and 1500 shots per spot were collected. For spectra calibration an external standard was used. Spectra were evaluated using the software flexAnalysis. TTR variants were expressed as relative amounts of the summed intensity of all observed TTR variants.RESULTS:The administration of NAC during a hemodialysis session resulted in a substantial increase of native TTR from median 15% (range 8.8-30%) to median 40% (37-50) and a reduction of S-cysteinylated TTR [51% (44-60) vs. 6.6% (2.4-10)]. Additionally the pronounced formation of a TTR-NAC adduct was detected. However, all these modifications seemed to be reversible. Additionally, in vitro incubation of plasma with NAC confirmed the in vivo results and indicated that changes in PTM pattern of TTR were a function of NAC concentration.CONCLUSIONS:We conclude that the interaction of N-acetylcysteine with proteins may explain altered protein functions due to beneficial modification of cysteine residues in hemodialysis patients.

AB - INTRODUCTION AND AIMS:The thiol antioxidant N-acetylcysteine (NAC) may mediate interactions with protein-associated cysteine residues, however, information on protein level in vivo are missing. Therefore, in the present study we aimed to analyze N-acetylcysteine-induced modifications of the protein transthyretin (TTR) in plasma from hemodialysis patients in a randomized, placebo-controlled study in vivo and after administration to plasma in vitro. TTR was selected due to its low molecular weight and the free cysteine residue in the polypeptide chain, which is known to be extensively modified by formation of mixed disulfides.METHODS:Plasma levels of TTR were determined by a non-commercial enzyme-linked immunosorbent assay (ELISA) using polyclonal rabbit anti-human TTR antibodies. Spectra of immunoprecipitated TTR were obtained using an AutoflexSpeed matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker-Daltonik, Bremen, Germany). The samples were analyzed in triplicates. For ionization, a Smartbeam-II laser was used and 1500 shots per spot were collected. For spectra calibration an external standard was used. Spectra were evaluated using the software flexAnalysis. TTR variants were expressed as relative amounts of the summed intensity of all observed TTR variants.RESULTS:The administration of NAC during a hemodialysis session resulted in a substantial increase of native TTR from median 15% (range 8.8-30%) to median 40% (37-50) and a reduction of S-cysteinylated TTR [51% (44-60) vs. 6.6% (2.4-10)]. Additionally the pronounced formation of a TTR-NAC adduct was detected. However, all these modifications seemed to be reversible. Additionally, in vitro incubation of plasma with NAC confirmed the in vivo results and indicated that changes in PTM pattern of TTR were a function of NAC concentration.CONCLUSIONS:We conclude that the interaction of N-acetylcysteine with proteins may explain altered protein functions due to beneficial modification of cysteine residues in hemodialysis patients.

M3 - Poster

ER -