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Department of Molecular and Cellular Physiology, LSU Health Sciences Centers, Shreveport, Louisiana, USA;
* Center for Cardiovascular Sciences, Albany Medical College, New York, USA; and
Department of Medicine, Division of Cardiology, Pulmonary Diseases and Angiology, Heinrich-Heine-University, D-40225 Duesseldorf, Germany
1Correspondence: Department of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130, USA. E-mail: mfeeli{at}lsuhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: nitrosothiols • nitrosamines • nitrosylheme • inflammation • plasma • red blood cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. RSNOs may be generated by reaction with either nitrosonium ions (NO+) formed from dinitrosyl–iron complexes (2)
or peroxynitrite (ONOO-) derived from the reaction of NO with superoxide (3
, 4)
. Alternatively, under anaerobic conditions RSNOs may be formed by direct interaction of NO with thiols in the presence of electron acceptors (5)
. The importance of the SNO group as a post-translational modification is evidenced by the multiplicity of recent reports indicating that proteins may be regulated by S-nitrosation of specific cysteine residues in vitro and in vivo (for review, see ref 6
). However, investigations into the validity of the hypotheses emerging about the physiological or pathophysiological role of specific nitros(yl)ated biomolecules are hampered by the availability of analytical techniques suited to allow reliable quantification of the extremely low levels of such species in vivo. Moreover, although RSNOs have been detected in various biological samples and are the focus of most recent studies, NO can interact either directly or indirectly with other biological targets (1)
, including amines and heme moieties. It is unclear, however, whether amine or heme moieties may also become nitros(yl)ated under the same experimental conditions and what the biological consequences of such protein modifications are.
Older colorimetric methods for nitrite and RSNO determination are subject to various interferences from proteins, suspended materials, and colored species; they lack sufficient sensitivity and often require elaborate clean-up procedures when working with biological media. Although EPR spectroscopy is a useful technique for determination of NO in metal complexes or hemeproteins (7)
and can be applied in vivo, it can only detect these and no other NO-related compounds. In addition, its sensitivity appears to be insufficient for nitrosyl detection under basal conditions (8)
. Several analytical procedures have been described for the determination of RSNOs and N-nitrosamines (RNNOs) based on chemical denitrosation and subsequent detection of the released NO by gas-phase chemiluminescence reaction with ozone (9)
. Though it has been demonstrated that NO can be released from RSNOs and RNNOs by illumination with UV light (10
11
12)
, in most procedures cleavage of the nitroso compounds is achieved by sample reduction with hydrogen bromide, iodide, or vanadium chloride. As the reducing properties of these solutions vary greatly, so do efficiency of reduction and selectivity. Most techniques available today were developed for in vitro quantification of select compounds and have never been validated for biological matrices. This includes the widely used Saville assay, which suffers from interference with proteins and reducing agents (13)
. This technique is based on the mercuric chloride (HgCl2) -induced cleavage of RSNOs to form nitrite and quantification of the latter by the classical Griess reaction (14)
. To complicate matters further and unbeknown to many researchers outside and inside the NO field, some sampling and sample processing techniques may even produce the species under investigation, adding to the confusion that surrounds the state of the art. In vivo, more than one type of nitros(yl)ated species may be formed at a time. For the most part, their identities will be unknown, in which case compound-specific techniques (15)
are of little help. The efficacy and selectivity of other methodological approaches, including the "biotin switch method" for RSNO identification (16)
and the use of antibodies against S-nitrosocysteine (17)
, still need to be proved and do not allow a quantitative assessment. Gas-phase chemiluminescence is exquisitely sensitive to trace quantities of NO, making it the method of choice when extremely low levels of nitroso species must be quantified in a complex matrix and sample volumes are limited. Here, we describe a sensitive analytic method for quantification of nitrite and nitroso compounds regardless of their structure and association with cellular constituents, in biological media. Using this technique, we find that besides RSNOs, endogenous RNNOs and possibly nitrosylheme species are present in all biological samples examined, but significant differences exist between different animal species. These results demonstrate that nitroso compounds other than RSNOs are formed in significant amounts in cells and tissues, the physiological role of which is unknown.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, 19)
, their stock solutions stored on ice in the dark and concentrations determined spectrophotometrically immediately before use. S-Nitrosohemoglobin (SNOHb) was prepared from oxyHb by trans-nitrosation (molar ratio oxyHb:CysNO 3:1) with the product revealing absorbance peaks at 540 and 575 nm, as described (20)
. SNOHb concentrations were determined using the Saville reaction (14)
. N-Nitroso-L-tryptophan was prepared either by nitrosation of L-tryptophan with gaseous NO2/N2O3 (by exposing aqueous tryptophan solutions to an NO/air mixture) or by reaction with excess acidified nitrite at room temperature (RT). All other RNNOs were obtained from commercial sources and prepared as 5 mmol/L stock in water, except nitrosobenzene and p-nitrosophenol, which were prepared in ethanol. Nitrosylhemoglobin (NOHb) was prepared from human deoxyHb with NO (NO:heme ratio 1:1 or 1:4) in phosphate buffer under argon. NOHb concentrations were determined spectrophotometrically (21)
whereas those of mononitrosylated hemoglobin (NOHb/oxyHb(1+3) hybrids) were determined using the methodology described in this paper, taking into account the recovery rate for fully nitrosylated Hb. Dilutions of either stock solution were made fresh in oxygen-free buffer and stored for 
3 h in the dark. Aqueous NO solutions were prepared as described (22)
and diluted in deoxygenated saline or phosphate buffer immediately before use. NO concentrations in these dilutions were determined by chemiluminescence under nonreducing conditions (injection into water), with nitrite contaminations being 2% of the NO concentration. NO sequestration studies were performed as described (23)
.
Biological samples
Arterial and venous blood of male Wistar rats, guinea pigs, C57BL/6J mice, and rhesus monkeys (Macaca mulatta) was collected in tubes containing N-ethylmaleimide (NEM)/EDTA (10/2 mmol/L) (24)
. Plasma was obtained by centrifugation for 10 min at 800 g and 4°C; erythrocytes were lysed 1:4 in water containing NEM/EDTA (10/2 mmol/L) and kept for 15 min on ice in the dark. Murine macrophage cells (J774.A1) were grown in T25 flasks to 
90% confluence using DMEM supplemented with 10% fetal calf serum. Cells were immunostimulated by treatment with either 0.1 or 1 µg/mL lipopolysaccharide (LPS; from S. typhosa) or a combination of LPS and 
-interferon (IFN; 10 U/mL). After 20 h of incubation cells were trypsinized, washed twice with PBS supplemented with NEM/EDTA (10/2 mmol/L) and resuspended in cold NEM/EDTA-containing PBS to a final density of 1 x 106 cells/mL. Cells were homogenized using a miniature glass homogenizer and the resulting homogenate was kept on ice in the dark for 15 min until analysis. In some experiments, the cell homogenate was subjected to centrifugation at 105,000 g and 4°C in order to separate the particulate fraction from the cytosol. Rats and mice were housed three/cage in a ventilated micro-isolator caging system and kept on a reversed 12/12 light/dark cycle. Animals were fed a standard rodent diet and allowed access to food and water ad libidum. Some animals were challenged by a single intraperitoneal (i.p.) injection of LPS (rats: 2 mg/kg; mice: 20 mg/kg) 18 h before organ removal. On the day of the experiment, animals were anesthetized with diethylether and killed by cervical dislocation. After thoracotomy, organs were flushed free of blood and perfused via the arterial inflow vessels with NEM/EDTA (10/2 mmol/L) supplemented PBS, which leads to efficient in situ alkylation of thiols and complexation of transition metals, preventing artifactual nitrosation and breakdown of already formed RSNOs. Organs were cut into small pieces and homogenized in ice-cold NEM/EDTA-containing phosphate buffer (50 mmol/L, pH 7.4; 5:1 volume/wet weight) using a Potter-Elvehjem homogenizer. Homogenates were kept on ice in the dark and used within 30 min of preparation. For stability studies, aliquots were frozen at -70°C and analyzed after 1 and 2 wk of storage.
Assay for quantification of nitrite and nitroso compounds
The concentration of nitrite and various nitroso species was determined after reductive cleavage by an iodide/triiodide-containing reaction mixture and subsequent determination of the NO released into the gas phase by its chemiluminescent reaction with ozone (O3). NO reacts with O3 to form nitrogen dioxide (NO2); a proportion of the latter arises in an electronically excited state (NO2*), which, on decay to its ground state, emits light in the near-infrared region and can be quantified by a photomultiplier (25)
. Provided O3 is present in excess and reaction conditions are kept constant, the intensity of light emitted is directly proportional to NO concentration (9)
. The reaction mixture, consisting of 45 mmol/L potassium iodide (KI) and 10 mmol/L iodine (I2) in glacial acetic acid, was kept at constant temperature in a septum-sealed, water-jacketed reaction vessel, continuously bubbled with nitrogen. The design of the reaction chamber was similar to a commercially available unit (Sievers, Boulder, CO), comprising a more efficient reflux condenser kept at 0°C and a larger chamber size to accommodate volumes up to 15 mL. The outlet of the gas stream was passed through a scrubbing bottle containing sodium hydroxide (1 mol/L; 0°C) in order to trap traces of acid and iodine before transfer into the detector (CLD 77AM sp, Eco Physics). To keep baseline noise at a minimum and achieve consistent results at trace level range, nitrogen flow must be kept absolutely constant throughout the entire measurement cycle. Best results were achieved using a pressure-controlled system where flow rates are determined by the diameter of the orifice of commercially available sample inlet tubes of different sizes (flow rates 50–350 mL/min). A pressure gauge was placed between the outlet of the scrubbing bottle and the detector inlet for continuous pressure monitoring and adjustment of purging gas flow (0±0.05 bar). Standards and sample aliquots (5–500 µL) were injected into the reaction mixture by gas-tight Hamilton syringes; NO signal output was sampled at 2 Hz. Peak integration was performed after signal smoothing to eliminate high-frequency noise and baseline correction using the ChromProcessor software. The analyzer was calibrated weekly using a 100 ppb mixture of NO in nitrogen. Calculated NO amounts were validated by injection of freshly prepared nitrite standards into a reaction mixture of 50 mg KI in 14 mL glacial acetic acid.
Data presentation and statistics
Data are presented either as original recordings or the means ± SE from n individual experiments with buffer and reagent blanks subtracted from each experimental value. Assay reproducibility was determined by comparison of the peak area from 10 repeated measurements of standards obtained on the same day (intra-day variation) or at 28 different days (inter-day variation) and expressed as percent coefficient of variation.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The assumption that NOx can react with biological tissues under physiological conditions has been further supported by experiments aimed at unraveling the phenomenon of photorelaxation, demonstrating that a depletable store of NO exists in vascular tissue (26)
. In preliminary studies carried out with different cell and tissue homogenates we observed that even in the complete absence of oxygen a certain amount of the NO added was not recovered, confirming earlier reports (23)
. More important, however, significant NO sequestration was observed even after chemical blockage of all titratable thiol groups (data not shown), providing indirect evidence for the existence of NOx-reactive sites other than thiols. With the exception of inhibition (e.g., cytochrome oxidase) and activation (e.g., guanylyl cyclase) by heme nitrosylation the potential biological consequences of NO-mediated post-translational protein modifications; e.g. via N-nitrosation of amines are largely unknown. This is due in part to the lack of appropriate analytical techniques to quantify the low concentrations of nitrosated species and differentiating between different compound groups in biological tissues and fluids. Thus, to gain further insight into the fate of NO and associated nitros(yl)ation reactions in vivo, a sensitive and specific analytical technique to quantify and discriminate between different nitroso species had to be developed.
Assay development and optimization
Earlier chemiluminescence-based techniques for quantification of RNNOs in food and beverages (27
, 28)
and more recent assays for RSNOs based on similar principles (24
, 29)
served as a starting point for development of the methodology we describe here. These techniques all rely on acidolytic or reductive denitrosation by hydrogen bromide, hydrogen iodide, or vanadium chloride solutions of different strengths, with a recent assay for quantification of SNOAlb (24)
containing copper ions in addition. What species other than RSNOs or RNNOs may be reduced to NO under the conditions of these assays has not been investigated in detail. Moreover, only a few methods have been validated for use in plasma and none for cell or tissue homogenates. Here, we describe a novel analytical technique that allows quantification of femtomol amounts of nitrite and nitroso compounds; describe what influence composition and volume of the reducing mixture, reaction temperature, and gas flow have on overall assay sensitivity, reaction yield, and peak size, and report on the results of our validation experiments and on the occurrence and nature of endogenous nitroso species in different biological matrices.
Redox-active mixtures of thiols with various transition metals at different molar ratios were systematically evaluated for their usefulness to detect small concentrations of SNOAlb in diluted human plasma. Similar to fruitless efforts by other laboratories with GSNO (29)
, we were unable to find reaction conditions that provided reproducible yields of NO formation, particularly at higher protein concentrations. More promising results, however, were obtained when Cu2+-salts were added to acidified bromide or iodide (I-) -containing solutions, resulting in mixtures resembling those described by our group earlier (24)
. Dissolution of crystalline iodine (I2) in aqueous solutions of I- produced reaction mixtures of comparable reducing strengths, making the addition of copper salts dispensable. I2 is known to form addition complexes with I-, known as triiodates (I3-). At acidic pH, these species exist largely in the form of their corresponding acids, HI and HI3. Since little information was available on electrochemical properties of the different redox couples (I-/I2, I-/I3-, I3-/I2) in iodide/iodine mixtures (30)
, predictions on how the ratio of iodine/iodide would affect the reduction potential of such solutions were impossible to make. Taking into account that biological samples contain variable amounts of redox-active compounds, which may result in the consumption of iodine or iodide, different strengths and molar ratios were investigated with regard to their efficacy to convert nitrite, GSNO, and SNOAlb to NO. The following reaction mixture was prepared: 38 mg I2 was added to a solution of 108 mg KI in 1 mL water and dissolved at RT under stirring. This I-/I3--containing stock was added to 13.5 mL glacial acetic acid, an excellent water-miscible solvent for halogen acids, to produce final concentrations of 45 mmol/L I- and 10 mmol/L I2. This mixture was compared to a 2x concentrated solution, a 1:2 and 1:10 dilution in acetic acid, and mixtures of different molar ratios of I-/I2 (2xI-, same I2 and 2xI2, same I-). The reaction mixture was maintained either at RT or heated to 40, 60, 70, and 90°C. Optimal results were obtained at 60°C. The use of higher temperatures accelerated the reduction process, producing narrower and higher peaks, but had little influence on NO yield. Whereas the overall reducing capacity of the reaction mixture decreased with decreasing I-/I3- content, decreasing the number of repeated injections possible, differences in peak areas between different reaction mixtures were small (±8%). However, results tended to be more variable at lower iodide concentration, and reduction of high concentrations of protein RSNOs proceeded noticeably slower. Importantly, the yield of NO production from nitrite or RSNOs did not change despite large variations in the ratio of I- over I3-. At 45 mmol/L I-/10 mmol/L I2 and 60°C, from hereon referred to as "standard conditions," the conversion of nitrite to NO was rapid and approached theoretical values (97–102%).
Calibration, reproducibility, and detection limit
With all samples the detector response was directly proportional to the amounts injected, thus sensitive to volume and concentration. No deviation from linearity was observed over the entire concentration range of 10 nmol/L to 100 µmol/L (r=0.9997, P<0.0001 for nitrite; n=7). Only peak areas, but not peak heights, correlate with the amounts of NO, nitrite or nitroso species injected, because at a given flow signal heights depend on injection speed, mixing, and prevailing reduction velocity. The detection limit for nitrite and readily reducible nitroso compounds (i.e., those producing sharp peaks) was 5 nmol/L at an injection volume of 100 µL (S/N ratio 3:1). A typical standard calibration curve for nitrite is depicted in Fig. 1
. Note that ultrapure water (18 M
/cm quality) produced a small signal when exposed to room air for more than a few minutes. This signal was found to be due to the hydrolysis of absorbed ambient N-oxides and was abolished by preincubation with sulfanilamide/H+ (see below). Assay sensitivity not only depends on denitrosation efficiency but on other factors, including gas flow, overall dead space, detector type, baseline noise, and background nitrite levels. With the system described here and an injection volume of 500 µL as little as 50 fmol NO can be detected in the form of nitrite and 100 fmol as protein-bound NO. Purging gas flow rates of 110–220 mL/min represent a good compromise between optimal sensitivity (low flow) and short analysis time (high flow). Precision at trace level range is better than 6%, and 20–40 samples can be analyzed per hour. Reproducibility, i.e., intra-day variability ranged from 2 to 7% with standards, depending on operator and pipette precision, and was typically 5–7% with biological samples, depending on the complexity of the matrix. The inter-day coefficient of variation was 4.7%.
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Discrimination between different nitroso compounds and nitrite
To differentiate between compound classes without having to change reaction solutions or conditions, samples can be pretreated with group-specific reagents before analysis. Biological samples are typically divided into three aliquots: one used for direct injection (nitrite+nitroso compounds), one for preincubation with sulfanilamide (total nitroso species), and another for preincubation with HgCl2/sulfanilamide (mercury-resistant nitroso compounds). As a general measure, samples are kept on ice in the dark to avoid photolytic and thermolytic decomposition.
Nitrite
Sulfanilamide has been used to efficiently remove nitrite from solutions treated with strong acids (24
, 31)
. The amount of nitrite in a given sample can be quantified by simple subtraction of the peak areas of sample aliquots pretreated with sulfanilamide from that of untreated aliquots (Fig. 2
A). For routine use, 10% (v/v) of a 5% solution of sulfanilamide in 1N HCl is added to the biological sample (final concentration 29 mmol/L) and incubated for 15 min at RT. Under these conditions, nitrite reacts with sulfanilamide to form a stable diazonium ion that is not converted to any appreciable extent to NO. After repeated injection of larger volumes of samples containing in excess of 0.5 mmol/L nitrite, however, gradual reduction of the diazonium ion was observed, resulting in an upward shift of the baseline. Although this does not affect peak areas of subsequently injected samples, it is best remedied by an exchange of the denitrosation solution.
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S-Nitrosothiols
The concentration of RSNOs present in a given sample can be conveniently quantified by calculation of the difference between the detector signal obtained in the presence of sulfanilamide/H+ (corresponding to total nitroso content) and that after pretreatment of the sample with 0.2% HgCl2 (final concentration 7.3 mmol/L) and sulfanilamide/H+. Incubation with HgCl2 results in cleavage of the S-NO bond without affecting peak shape or recovery of nitrite or NO and forms the basis for the widely used Saville assay (14)
. Time course studies with GSNO and SNOAlb revealed that at physiological pH, a >20 min incubation period is required for complete S-NO cleavage. No difference was found between sequential pretreatment with HgCl2, followed by sulfanilamide/H+ and coincubation with both agents under acidic conditions. Hence, a 30 min incubation with the combined HgCl2/sulfanilamide/HCl reagent was adopted for routine RSNO analysis.
Other nitroso species
The mercury-resistant part of the detector signal (i.e., the peak remaining after preincubation with HgCl2/sulfanilamide/H+) indicates the presence of RNNOs or metal nitrosyls such as NO-heme species in the sample. Extensive further validation experiments with aqueous standards confirmed that none of the species tested showed a >5% sensitivity toward mercury-induced cleavage under these conditions (Table 1
). The power of the above approach is demonstrated by comparison of the signals obtained on injection of SNOAlb and pNOAlb, a derivative that in addition to the cysteine thiol is nitrosated at a tryptophan residue (32)
and possibly elsewhere in the molecule. As shown in Fig. 2B
, SNOAlb is fully Hg2+-cleavable whereas pNOAlb has a Hg2+-resistant part due to the presence of nitrosated NH2 residues. Using the same approach we found that a significant portion of all "SNOHb" preparations obtained by trans-nitrosation of oxyHb with CysNO (20)
is HgCl2-resistant, indicating that this route of synthesis produces substantial amounts of species other than the expected RSNO (33)
.
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Nitroso species detected and mechanism of denitrosation
A major goal of the present study was not only to characterize what species are reduced to NO by our reaction mixture, but also to find a means to differentiate between compound classes likely to be present in biological tissues and fluids. Thus, besides the S-nitroso compounds SNOAlb, GSNO, CysNO, and SNAP, a series of different N-, O-, and C-nitroso compounds and iron nitrosyls were tested. All but three compounds were found to produce a signal under standard assay conditions (see inset of Fig. 3
for representative tracings of select compounds). Typical NO yields for RSNOs approached 100% for low molecular weight compounds and 75–80% for protein RSNOs (see Table 1
). These values compare favorably to those obtained with other reductive techniques (29
, 34)
or photolytic cleavage (11)
. Although the reduction of most N-nitroso compounds to NO was markedly slower than that of RSNOs, overall NO yields were in the range of 67–105%. In agreement with earlier findings on the relative resistance of nitrosamides toward denitrosation by hydrogen bromide (35)
, lower reduction yields were obtained with N-nitrosourea. Whereas isoamyl nitrite was readily reduced to NO, nitrosobenzene and p-nitrosophenol did not give a signal at concentrations up to 100 µmol/L. The lack of detection of nitroprusside was unexpected as this compound is a known NO donor that rapidly decomposes in most biological tissues. The denitrosation yield for NOHb was in the same range as that for SNOAlb. Thus, the iodine/iodide assay allows quantification of S-, N-, and O-nitroso compounds as well as nitrosylheme species.
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The reaction mechanism for RSNO cleavage proposed by Samouilov and Zweier (29)
—I2-mediated oxidation to form thiyl radicals (RS.) and nitrosonium ions (NO+) followed by I--mediated reduction of NO+ to NO—cannot account for the denitrosation of RNNOs and nitrosylheme species. Moreover, the concentration of free I2 is low in the presence of excess HI due to formation of triiodic acid (HI3). Formation of a cage-like cyclic intermediate with elimination of the NO group as nitrosyl triiodide (NOI3) and subsequent reduction of NO+ by HI may account for the denitrosation of some but not all of the nitroso compounds tested. A similar mechanism was proposed earlier for the denitrosation of RNNOs by hydrogen bromide (36)
. Further mechanistic investigations are required to elucidate the nature of the underlying reaction mechanism, but this was beyond the scope of the present investigation.
Potential assay interferences
Using our standard conditions, 
10 repeated injections of 10 µmol/L SNOAlb or GSNO could be made using the same reaction mixture without a noticeable change in recovery or peak shape. Trace level quantification of nitroso species (<100 nmol/L) was not influenced by prior injection of high amounts of nitrite or SNOAlb. Dilution of the reaction mixture with 1 mL water or sulfanilamide-preincubated nitrite standards did not affect peak shape or sensitivity either.
Protein and heme
Repeated injections of up to 2 mL of an albumin-containing solution (4g/dL) had no effect on NO yield from nitrite. However, protein-rich samples containing additional redox-active constituents may lead to rapid exhaustion of the denitrosation reagent. This is easily recognized by the peak broadening occurring under these conditions and may be conveniently checked by injection of nitrite standards between unknowns. Samples rich in hemeproteins may lead to peak broadening of subsequent samples. This effect is due to NO capture; i.e. trapping of the NO cleaved off another nitroso moiety by the heme. However, peak areas of nitrite standards and biological samples are usually within 95–99% of those in the absence of heme unless concentrations approach millimolar levels (Fig. 3)
. Preincubation with cyanide (10 mmol/L, 20 min) has no effect on peak broadening.
Redox-active compounds
Possible interference with endogenous redox-active agents was tested by addition of ascorbate, NADPH, NADH, FAD, or GSH (all 1 mmol/L) to SNOAlb (10 µmol/L) in buffer. Spiked samples were vortexed for 30s and measured immediately. None of the compounds tested produced a false positive signal or had an inhibitory influence on the denitrosation process (peak areas were within±5% of those in the absence of the redox compounds).
Cross sensitivity
Susceptibility of the assay to give false positive signals was assessed by investigation of potential cross-reactions with non-nitroso species, including inorganic nitrate (NO3-), the O-nitro compounds isopentyl nitrate and nitroglycerin, and the nitro derivatives NG-nitro-L-arginine methyl ester and 3-nitrotyrosine. Although some compounds contained traces of nitrite, none produced a signal at concentrations 1 mmol/L (50 µL inject. volume), indicating that the denitrosation mixture reduces neither inorganic nitrate nor nitrate esters or nitro derivatives.
Prevention of artifactual nitrosation and degradation of nitroso compounds
Already at physiological pH, but particularly on acidification, RSNOs are formed artifactually when reduced sulfhydryl (-SH) groups and nitrite are present together at any one time (18)
. In addition, amine nitrosation may be catalyzed by thiols (37)
. Although thiols and nitrite are present in most biological samples, the significance of such reactions seems to have largely been ignored. Artifactual nitrosation can be prevented by nitrite removal and blockade of SH groups. Even though size exclusion chromatography (using e.g., Sepadex-G25 columns) is an option for samples containing high molecular weight compounds, nitrosated low molecular weight species will also be removed and separation from nitrite is often incomplete. Therefore, selective removal of nitrite in solution is preferable and less time-consuming. Since nitrite scavengers generally work at low pH only and acidification favors nitrosation, thiol alkylation before nitrite removal is an absolute requirement. Competition experiments with nitrite and GSH (not shown) revealed that sulfanilamide and azide are kinetically superior to all other scavengers and, at high concentration, can compete with low molecular weight thiols for nitrite. Nevertheless, even with the fastest and most efficient nitrite scavengers, sample pretreatment with a sulfhydryl-blocking agent is mandatory to prevent artifactual formation of trace quantities of RSNOs in biological samples (see below). We advocate the use of excess NEM over other thiol-alkylating agents (such as iodoacetamide) as it readily penetrates cell membranes and can be added to the washing or perfusion buffer during cell/tissue harvesting. Another potential problem relates to the limited stability of endogenous RSNOs during cell/tissue homogenization, triggering degradation of previously compartmentalized compounds after exposure to reduced thiols or ascorbate. The extent to which this occurs depends on the redox environment and prevailing concentrations of either reactant. Thiols are blocked using the same strategy whereas ascorbate-mediated RSNO breakdown is prevented by complexation of transition metals using EDTA or DTPA. Hence, a protocol using 15 min preincubation with 10 mmol/L NEM and 2 mmol/L EDTA was employed.
Detection of nitrosated compounds in cultured cells, biological fluids, and tissues
A major aim of this study was to develop an analytical tool that allows sensitive and reliable quantification of nitroso compounds in biological samples. The ultimate test of whether the "standard assay conditions" described above would hold promise in a complex biological matrix therefore was to apply them to authentic cell/tissue samples and body fluids. Results described in the following demonstrate that this goal has been achieved in that nitrosated species of endogenous origin were readily detected under basal and stimulated conditions. Besides RSNOs, all samples unexpectedly contained RNNOs and/or nitrosylhemes.
First, recovery experiments were carried out by spiking rat plasma, liver homogenate, and J774 lysates with GSNO or SNOAlb to produce final concentrations of 1 and 5 µmol/L; peak areas obtained in the biological media were compared to those obtained in buffer. Recoveries approached 96% and 87% for 1 and 10 µmol/L SNOAlb and 102% and 91% for 1 and 10 µmol/L GSNO. The formation of nitroso compounds in response to endogenously generated NO was then investigated using the murine macrophage cell line J774. Unstimulated J774 cells produced negligible amounts of nitrite within 24 h, confirming the lack of constitutive NOS activity. In contrast, after iNOS induction, cells produced large amounts of NO. Figure 4
A depicts the time course of NO formation after stimulation with 1 µg/mL LPS as monitored by the accumulation of nitrite in the cell culture medium. NO production started between 4 and 6 h and was maximal 12 h after stimulation. NO formation was associated with the nitrosation of cellular constituents, which peaked at the time of maximal NOS activity, declining thereafter (Fig. 4B
). The decrease in cellular nitroso content was not due to loss of cells by detachment, suggesting that nitrosation was rapidly reversible with decreasing NO production. Although LPS stimulation of native J774 cells resulted in robust levels of NOS activity, even higher responses were elicited in cells primed with -interferon (Fig. 5
A). NO-mediated nitrosation products were found not only in the cytosol but also in the particulate cell fraction (Fig. 5B
), demonstrating that water-insoluble, protein-bound nitroso species are readily detected by our assay. Comparative experiments carried out in the presence and absence of NEM revealed that thiol alkylation is an absolute requirement for preventing artifactual nitrosation in biological samples. The effect is highest in samples containing high micromolar thiol exemplified by the >100% increase in apparent RSNO levels in the cytosol of stimulated macrophages when NEM was omitted from the homogenization buffer (Fig. 5B
).
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Part of the signal in particulate and cytosolic fraction was mercury-stable, indicating that NO was bound to sites other than thiols. Triggered by this observation, investigations into the sample sensitivity to HgCl2 were extended to organ homogenates of rats challenged with a relatively low dose of LPS, erythrocyte lysates and plasma of untreated rats, guinea pigs, mice, and monkeys. The results obtained demonstrate that not every nitroso signal is due to the presence of RSNOs and that other reactive sites exist that can effectively compete with thiols for endogenous NOx. Alternatively, these species may be the product of S
N trans-nitrosation reactions. Immunostimulation with LPS in vivo is known to result in a systemic inflammatory response accompanied by the induction of iNOS in all major organ systems, including the liver (38)
. We therefore investigated whether the increase in NO production under these conditions results in tissue nitrosation in laboratory rats. Indeed, on LPS stimulation, levels of RSNOs and mercury-stable nitroso species increased to a different extent in all organs investigated (Fig. 6
). Similar results were obtained in mice and guinea pigs (not shown). To the best of our knowledge, this is the first demonstration of in vivo nitrosation in response to iNOS induction throughout the organ system. In all samples, nitroso levels gradually decreased over 2 wk of storage at -80°C, demonstrating that analyses should be performed with fresh samples to avoid underestimation.
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Further investigation in the blood of untreated experimental animals showed that plasma and erythrocytes of all species contained nitrite, RSNOs, and a mercury-resistant nitroso component of unknown identity (Fig. 7
). The latter corresponded to 20–75% of the total nitroso content, suggesting that physiological RNNO levels may be considerably higher than RSNO levels in some species. SNOHb levels in erythrocytes from arterial and venous rat blood amounted to 288 ± 25nmol/L and 74 ± 22nmol/L (n=5–8), confirming earlier reports (20)
and demonstrating that reliable measurements of nitrosated compounds can be made even in the presence of high hemoglobin concentrations. No apparent arterial/venous differences were seen in plasma RSNO levels of any species investigated. Comparative measurements of RSNOs and mercury-stable nitroso compounds in different animals revealed marked species differences with rather high levels in rat, mouse, and guinea pig erythrocytes, but a complete lack of nitroso species in erythrocytes from rhesus monkeys. This striking difference between rodents and primates may be due to differences either in hemoglobin structure and function or in the reducing capacity of their red blood cells.2
Whatever the cause, these differences are important to keep in mind when comparing data between different animal species and extrapolating results obtained in experimental animals to humans. Taken together, our findings suggest that NO generation in vivo leads to concomitant formation of RSNOs, nitrosamines, and nitrosylhemes, with extent and ratio varying between species. This underscores the difficulty in comparing data from different animal models and extrapolating results obtained in experimental animals to human physiology.
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Possible nature of nitroso species present in biological samples
Although our assay offers a means to distinguish nitrite and RSNOs from other nitroso species, we were unable to find a way to differentiate N-nitroso compounds from nitrosylhemes. With the abundance of hemeproteins such as cytochrome P450, cyclooxygenase, and peroxidases in tissues and their known high affinity for NO, heme moieties appear to be obvious acceptors for endogenous NO in vivo. Thus, part of the mercury-resistant signal observed in stimulated macrophages and organ homogenates may be due to the presence of nitrosylhemes rather than RNNOs. To discriminate between these species, a method suited to selectively cleave off NO from the heme would be required. To date no such technique exists. Gladwin and co-workers (39)
recently proposed the use of ferricyanide/cyanide to selectively cleave NO from hemoglobin before analysis by a methodology similar to the one presented here. This approach relies on the classical Evelyn and Malloy technique of hemoglobin quantification in blood based on the oxidation of oxyHb to methemoglobin and measurement of the cyano-metHb complex by spectrophotometry (40)
. However, we find that whereas NOHb/oxyHb(1+3) hybrids, i.e., compounds where only one of the four hemes are occupied by NO, are oxidized to metHb, this is not accompanied by a loss of NO (T. Rassaf and M. Feelisch, unpublished observations). An unusually stable NOHb in valency hybrids has been described before (41)
. This has potentially important implications for studies of NO bound to hemoglobin in red blood cells and warrants further investigation.
The likelihood of picking up O-NO species in biological samples is diminished by trans-nitrosation reactions with thiols to form RSNOs (which are minimized by NEM pretreatment) and decomposition to nitrite during preincubation of samples with a nitrite scavenger under acidic conditions. The presence of endogenous dinitrosyl–iron complexes (DNICs) is similarly difficult to assess. To the best of our knowledge, attempts to detect these species by EPR spectroscopy under basal conditions have been unsuccessful. Whether such species are reduced under our assay conditions has not been specifically investigated. The rapid interconversion of DNICs and RSNOs (42)
makes it appear likely that these compounds decompose during tissue homogenization and are picked up as RSNO. The presence of C-nitroso species in biological samples cannot be excluded yet, as these species do not produce a signal under our assay conditions, although thiols, hemes, and amines are likely to out-compete possible C-nitrosation targets from a quantitative and kinetic point of view. Thus, a definitive characterization of the nature of nitroso species in cells and tissues awaits further development of techniques suited to selectively cleave defined compound classes without affecting others in the same biological matrix. In the meantime, a careful assessment of the formation and dynamics of S- and N-nitrosated, as well as heme-nitrosylated species, under different experimental conditions should provide important new insights into the role and significance of post-translational nitrosation reactions in physiology and pathophysiology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 15, 2002. Accepted for publication July 22, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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