Methods
A variety of different spectrophotometric methods have been developed to quantify nitric oxide (NO)-derived metabolites such as nitrate and nitrite. Some of these methods measure the formation of colored complexes such as azo dyes (e.g., Griess reaction), whereas other methods measure the formation of fluorometric compounds. Virtually all of these methods require the rapid and spontaneous decomposition...
NO − 2 and NO − 3 are quantitatively reduced to NO for chemiluminescence detection in mildly acidic vanadium(III) solution. Nanomolar detection limits are obtained with this method. Variation in application of the method allows for nitrite-only analysis, nitrite plus nitrate, or nitrite and nitrate in the same sample using a temperature-cycling routine. Analysis rates of 2...
Quantitative studies of the release of NO in vivo are difficult due to its short half-life. Consequently, most investigators have assayed nitrite and/or nitrate because these are relatively stable markers of NO production. Although various methods have been developed, all are limited to some extent by the degree of sample handling required. To overcome this we have developed a simple capillary electrophoresis...
Peroxynitrite is the product of the nearly diffusion limited reaction between two free radicals, nitric oxide and superoxide. Peroxynitrite is a relatively long-lived, highly reactive species that can undergo harmful secondary oxidations of key cellular components. The reactivity of peroxynitrite is highly pH dependent and is influenced by the presence of metal, thiols, and bicarbonate. The products...
This report will summarize some kinetic investigations of different reactions involving nitric oxide (NO) that are potentially relevant to biological chemistry. Such studies should provide a quantitative understanding of possible pathways in which NO may participate. On the basis of this information, it should be possible to determine the most significant pathways among the multitude of chemical trajectories...
Nitric oxide (NO) is a vasodilator, neurotransmitter, and inflammatory mediator which is unstable at physiologic O 2 tensions. NO′s susceptibility to oxidation accounts for its short biological half-life in vivo and greatly complicates its measurement. Measuring NO requires an understanding of the redox reactions and partitioning that determine its survival and distribution. In aqueous solutions...
The use of electron magnetic resonance (EMR) spectroscopy, also known as electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy, to detect and quantify the paramagnetic molecule nitric oxide, NO, in model systems, cells, and living organisms is reviewed. The physical basis for EMR spectroscopy and the information available from this method are presented. Since free NO...
Nitric oxide (NO) is a colorless gas that contains an unpaired electron which is delocalized over the nitrogen and oxygen atoms. This free radical has been identified as a biomolecule with important physiological functions. Nitric oxide has been shown to be important in the homeostatic regulation of the cardiovascular and central nervous systems as well as an important modulator of the immune system...
Two convenient colorimetric methods are described for the determination of nitric oxide (NO) concentration in neutral aqueous solutions. Both techniques take advantage of the fact that nitrosating and oxidizing intermediates are generated in the course of the NO/O 2 reaction. In the first method, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) is oxidized to the corresponding cation radical,...
In this article we discuss methodology for quantitation of nitrite and nitrate, end products of the biological synthesis of nitric oxide from L-arginine. Both nitrite and nitrate can be measured, with accuracy to the low micromolar range, using bacterial nitrate reductase coupled to the Griess reaction. Methods for preparing nitrate reductases from Escherichia coli and Pseudomonas oleovorans are outlined...
The use of electrochemical methods for the detection of various biological substances has provided real-time measurements giving insights into their roles in various physiological functions. The discovery that nitric oxide (NO) plays critical regulatory roles in a variety of physiological processes has prompted development of analytical techniques for the detection of this diatomic molecule in biological...
Nitrotyrosine has been found in the urine of humans with no known exposure to exogenous nitrating agents. We have shown that peroxynitrite, a nitrating agent formed by the near diffusion-limited reaction of nitric oxide with superoxide, is formed by activated macrophages. Using an antibody which recognizes nitrated tyrosine residues in proteins, we have obtained immunohistological evidence for nitration...
There is a growing body of both clinical and experimental data that demonstrates that activation of the immune system, whether locally or systemically, is associated with an increased production of nitric oxide (NO) as measured by increases in plasma and/or urinary levels of nitrate (NO − 3 ) and nitrite (NO − 2 ). Because NO may mediate some of the tissue injury and dysfunction...
Nitric oxide can be rapidly extracted from dilute aqueous solutions into the gas phase by diffusion through hydrophobic membranes, permitting direct measurement of nitric oxide and observation of its reactions by a commercial chemiluminescent nitric oxide detector. Nitric oxide is a hydrophobic gas, crossing membranes much as oxygen and carbon dioxide do in blood gas analyzers. A loop of hydrophobic...
While rarely used alone for quantification of nitric oxide synthesis, bioassay remains an important and sensitive tool for demonstrating the release of biologically active nitric oxide from isolated cells or tissues and is an inexpensive, readily accessible technique. In this article, we have reviewed the use of the platelet aggregation bioassay for detection of nitric oxide release. The preparation...
Nitric oxide (NO) released from cells or other sources is very rapidly oxidized to nitrite in the presence of oxygen. This article describes a procedure utilizing 1,1′-dimethylferrocene as the reducing agent for nitrite to nitric oxide and its subsequent determination by chemiluminescence. The introduced method allows the determination of nitric oxide together with nitrite. The total nitric oxide...
It is now well established that nitric oxide (NO) is both a critical bioregulatory agent essential to normal physiological function and a potentially toxic species. The primary goal of our NO research program is to investigate the possibility that exposure to NO might increase cancer risk. After reviewing some initial chemical and cella culture experiments from our laboratories suggesting that this...
A method for measuring repair in extracts of human cells of DNA containing mismatched bases is described. Wild-type and mutant M13mp2 phage derivatives are used to prepare a double-stranded DNA substrate that contains a nick in the (-) strand and mismatched bases in the lacZα-complementation gene. Introduction of this heteroduplex DNA into an Escherichia coli strain defective in mismatch repair yields...
We present a sensitive, nonradioactive method for DNA double-strand break (DSB) quantification in the yeast Saccharomyces cerevisiae (or any other organism with linear chromosomes =<9 Mb). Our technique allows studies of induction, repair, and local distribution of DSB induced by radiation and chemicals or of physiological origin. DSBs cause a loss of intact chromosomal length DNA and a simultaneous...