Determination of s-nitrosothiols in biological fluids by chemiluminescence.

Nagababu Enika, Joseph M. Rifkind

Research output: Contribution to journalArticle

Abstract

S-nitrosothiols present in nanomolar concentrations in cells and body fluids play an important role in vasodilation, in preventing platelet aggregation, leukocyte adhesion, and for cellular signaling. However, because of the low levels of s-nitrosothiols and interference with other nitric oxide species, reliable assays that measure both high molecular weight and low molecular weight s-nitrosothiols in plasma and red blood cells red blood cells have been difficult to develop. We have previously developed a sensitive method using Cu(II)-ascorbic acid Cu(II)-ascorbic acid at a neutral pH, which was specific for s-nitrosothiols without interference of nitrite or other NOx species. However, due to neutral pH foaming, this method was not suitable for determinations in plasma or red blood cells with high protein content. This method has now been modified by using copper (II) chloride (CuCl(2)) and ascorbic acid in glacial acetic acid. The low pH solves the foaming problem. However, protonation of nitrite under acidic conditions facilitates the formation of s-nitrosothiols. For this method to specifically measure s-nitrosothiols in the sample, the unreacted thiols are blocked by reacting with N-ethylmaleimide and nitrite is blocked by reacting with acidified sulfanilamide before being analyzed by chemiluminescence. Using this method, s-nitrosothiols have been determined in the range of 2 nM to 26 nM (mean ± SE = 10.18±2.1) in plasma and up to 88.1 nM (mean ± SE = 51.27 ± 10.5) in red blood cells.

Original languageEnglish (US)
Pages (from-to)27-37
Number of pages11
JournalMethods in molecular biology (Clifton, N.J.)
Volume704
StatePublished - 2011
Externally publishedYes

Fingerprint

Luminescence
Nitrites
Erythrocytes
Ascorbic Acid
Molecular Weight
S-Nitrosothiols
Ethylmaleimide
Body Fluids
Platelet Aggregation
Sulfhydryl Compounds
Vasodilation
Acetic Acid
Nitric Oxide
Leukocytes
Proteins

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Determination of s-nitrosothiols in biological fluids by chemiluminescence. / Enika, Nagababu; Rifkind, Joseph M.

In: Methods in molecular biology (Clifton, N.J.), Vol. 704, 2011, p. 27-37.

Research output: Contribution to journalArticle

@article{d146e7b9264c45f19309cbff240debab,
title = "Determination of s-nitrosothiols in biological fluids by chemiluminescence.",
abstract = "S-nitrosothiols present in nanomolar concentrations in cells and body fluids play an important role in vasodilation, in preventing platelet aggregation, leukocyte adhesion, and for cellular signaling. However, because of the low levels of s-nitrosothiols and interference with other nitric oxide species, reliable assays that measure both high molecular weight and low molecular weight s-nitrosothiols in plasma and red blood cells red blood cells have been difficult to develop. We have previously developed a sensitive method using Cu(II)-ascorbic acid Cu(II)-ascorbic acid at a neutral pH, which was specific for s-nitrosothiols without interference of nitrite or other NOx species. However, due to neutral pH foaming, this method was not suitable for determinations in plasma or red blood cells with high protein content. This method has now been modified by using copper (II) chloride (CuCl(2)) and ascorbic acid in glacial acetic acid. The low pH solves the foaming problem. However, protonation of nitrite under acidic conditions facilitates the formation of s-nitrosothiols. For this method to specifically measure s-nitrosothiols in the sample, the unreacted thiols are blocked by reacting with N-ethylmaleimide and nitrite is blocked by reacting with acidified sulfanilamide before being analyzed by chemiluminescence. Using this method, s-nitrosothiols have been determined in the range of 2 nM to 26 nM (mean ± SE = 10.18±2.1) in plasma and up to 88.1 nM (mean ± SE = 51.27 ± 10.5) in red blood cells.",
author = "Nagababu Enika and Rifkind, {Joseph M.}",
year = "2011",
language = "English (US)",
volume = "704",
pages = "27--37",
journal = "Methods in Molecular Biology",
issn = "1064-3745",
publisher = "Humana Press",

}

TY - JOUR

T1 - Determination of s-nitrosothiols in biological fluids by chemiluminescence.

AU - Enika, Nagababu

AU - Rifkind, Joseph M.

PY - 2011

Y1 - 2011

N2 - S-nitrosothiols present in nanomolar concentrations in cells and body fluids play an important role in vasodilation, in preventing platelet aggregation, leukocyte adhesion, and for cellular signaling. However, because of the low levels of s-nitrosothiols and interference with other nitric oxide species, reliable assays that measure both high molecular weight and low molecular weight s-nitrosothiols in plasma and red blood cells red blood cells have been difficult to develop. We have previously developed a sensitive method using Cu(II)-ascorbic acid Cu(II)-ascorbic acid at a neutral pH, which was specific for s-nitrosothiols without interference of nitrite or other NOx species. However, due to neutral pH foaming, this method was not suitable for determinations in plasma or red blood cells with high protein content. This method has now been modified by using copper (II) chloride (CuCl(2)) and ascorbic acid in glacial acetic acid. The low pH solves the foaming problem. However, protonation of nitrite under acidic conditions facilitates the formation of s-nitrosothiols. For this method to specifically measure s-nitrosothiols in the sample, the unreacted thiols are blocked by reacting with N-ethylmaleimide and nitrite is blocked by reacting with acidified sulfanilamide before being analyzed by chemiluminescence. Using this method, s-nitrosothiols have been determined in the range of 2 nM to 26 nM (mean ± SE = 10.18±2.1) in plasma and up to 88.1 nM (mean ± SE = 51.27 ± 10.5) in red blood cells.

AB - S-nitrosothiols present in nanomolar concentrations in cells and body fluids play an important role in vasodilation, in preventing platelet aggregation, leukocyte adhesion, and for cellular signaling. However, because of the low levels of s-nitrosothiols and interference with other nitric oxide species, reliable assays that measure both high molecular weight and low molecular weight s-nitrosothiols in plasma and red blood cells red blood cells have been difficult to develop. We have previously developed a sensitive method using Cu(II)-ascorbic acid Cu(II)-ascorbic acid at a neutral pH, which was specific for s-nitrosothiols without interference of nitrite or other NOx species. However, due to neutral pH foaming, this method was not suitable for determinations in plasma or red blood cells with high protein content. This method has now been modified by using copper (II) chloride (CuCl(2)) and ascorbic acid in glacial acetic acid. The low pH solves the foaming problem. However, protonation of nitrite under acidic conditions facilitates the formation of s-nitrosothiols. For this method to specifically measure s-nitrosothiols in the sample, the unreacted thiols are blocked by reacting with N-ethylmaleimide and nitrite is blocked by reacting with acidified sulfanilamide before being analyzed by chemiluminescence. Using this method, s-nitrosothiols have been determined in the range of 2 nM to 26 nM (mean ± SE = 10.18±2.1) in plasma and up to 88.1 nM (mean ± SE = 51.27 ± 10.5) in red blood cells.

UR - http://www.scopus.com/inward/record.url?scp=79955672552&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79955672552&partnerID=8YFLogxK

M3 - Article

C2 - 21161627

AN - SCOPUS:79955672552

VL - 704

SP - 27

EP - 37

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -