Nitric Oxide Determinations: Much Ado About NO•-Thing?

The nitric oxide radical (NO•) is an important mediator of both physiological and pathophysiological processes. NO• is produced by nitric oxide synthase (NOS; EC 1.14.13.39), an enzyme that exists in three isoforms encoded by distinct genes (1)(2). All isoforms of NOS catalyze the conversion of l-arginine into citrulline and NO•. In this reaction, which requires oxygen and NADPH, a guanidino-nitrogen atom of l-arginine is incorporated into NO•. Neuronal NOS (type I, nNOS) and endothelial (type III, eNOS) are Ca2+- and calmodulin-dependent constitutive NOS isoforms. nNOS has a function in neurotransmission. NO• produced by eNOS is identical to endothelium-derived relaxing factor and is the principal signal for relaxation of vascular smooth muscle cells. In addition, NO• produced by the endothelium has antithrombotic actions. Thus, eNOS and nNOS isoforms have important functions under normal conditions. They are present intracellularly, are rapidly activated by intracellular Ca2+ fluxes, and produce small quantities of NO•. Inducible NOS (iNOS, type II) is not expressed under normal conditions. iNOS is induced by cytokines and (or) endotoxin during inflammatory and infectious processes and produces abundant amounts of NO• for extended periods. iNOS can be induced in many cell types, including hepatocytes, macrophages, neutrophils, smooth muscle cells, and chondrocytes. Induction of iNOS requires de novo protein synthesis. NO• produced by iNOS has antimicrobial activity and may be involved in killing tumor cells. As such, it is part of the nonspecific host defense system. Increased expression of iNOS has been demonstrated in a wide range of disorders, including sepsis, asthma, rheumatoid arthritis, atherosclerotic lesions, tuberculosis, inflammatory bowel disease, Helicobacter pylori-induced gastritis, allograft rejection, Alzheimer disease, and multiple sclerosis (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). It has been postulated that excessive and continuous production of NO• is responsible for at least part of the symptoms in these disorders. Considering the normal functions of NO• and the wide range of pathological conditions in which it has been implicated, interest in measuring NO• production has, not surprisingly, received enormous attention recently (15). Many different methods for determining NO• or its metabolites nitrite and nitrate have been reported. The most relevant for the clinical chemist are briefly discussed here. •determination. Several methods for “real-time” NO• production have been reported. These methods detect ongoing NO• production in viable biological samples such as tissue explants, biopsies, or isolated cells (e.g., leukocytes or peritoneal exudate cells). Electrochemical methods use NO•-sensitive microsensors, e.g., Clark-type and porphyrinic electrodes (15)(16)(17). Recently, a microsensor based on a porphyrinic electrode was applied in vivo to humans (17). NO• can also be detected by chemiluminescence (15)(18) in an assay based on the reaction of NO• with ozone to yield •NO2 and light. NO• produced by viable biological samples reacts with ozone in the gas phase in a sealed compartment (headspace gas method). The emitted light can be detected with a photomultiplier tube. The same principle is used to measure NO• in exhaled air. Finally, NO• can be determined by a spectrophotometric assay in which NO• reacts with ferrous oxyhemoglobin (HbO2) to yield nitrate and methemoglobin (15)(18)(19). The formation of methemoglobin can be monitored by measuring the increase in absorbance at 401 nm. This method is suitable for in vitro and ex vivo measurements of NO• in viable biological samples. The advantage of these methods is that actual NO• formation is determined. Their disadvantages are technical complexity and the requirement for viable biological samples. Accordingly, these methods are not suitable for body fluids devoid of cells. In addition, application of this method in vivo is difficult and has been described for only the electrochemical sensors (17). The NO• radical is rapidly metabolized into the stable end-products nitrite and nitrate. In most body fluids, including plasma, most of the nitrite is converted to nitrate; thus determination of nitrite alone is meaningless (20). The most commonly used nitrite assay is based on the Griess diazotization reaction, which is specific for nitrite and does not detect nitrate. Therefore, nitrate in samples must first be reduced to nitrite; subsequent nitrite determination thus yields the total nitrite + nitrate concentration of the sample. Reduction of nitrate to nitrite can be performed by treating the samples with nitrate reductase (20)(21), copperized cadmium (22) or hydrazine (23). Nitrite and nitrate can also be determined in body fluids by HPLC (24), capillary electrophoresis (25)(26), or anion-exchange chromatography (27). Nitrite and nitrate ions are then detected by UV absorbance or conductivity. In this issue of Clinical Chemistry, Yang et al. (28) describe a very sensitive method for chemiluminescent detection of nitrite and nitrate. Nitrite and nitrate are first reduced to NO•, which is then reacted with ozone and detected by chemiluminescence in the gas phase as already described. The authors evaluated their method with respect to reproducibility, recovery, and reduction conditions. The importance of this method is the application of chemiluminescence to the detection of nitrite and nitrate, making this method more sensitive than most others currently used to measure nitrite and nitrate. Methods for determining nitrite and nitrate are applicable to both fresh and archived body fluids such as plasma, serum, urine, bile, synovial fluid, sputum, and cerebrospinal fluid. Advantages are the potential for automation and high throughput. A drawback is that such methods also detect nitrate derived from non-NO• sources such as diet. The exact contribution of diet or other non-NO• sources is uncertain. Fasting for 12 h is reported to reduce concentrations of plasma nitrate and nitrite by 50%, and in fasted healthy volunteers ∼90% of plasma nitrite and nitrate is derived from NOS-derived NO• (29)(30). Another disadvantage is that determination of nitrite and nitrate does not demonstrate ongoing NO• production. Considering that the half-life of nitrate in plasma is ∼1.5 h (31), increased concentrations of nitrate in plasma at least indicate recent NO• production. In this technique, [15N]-guanidino-labeled l-arginine is administered to subjects (29)(30). As catalyzed by NOS, the 15N at the guanidino position of l-arginine ends up in nitrite and nitrate. Enrichment of nitrite and nitrate with 15N can be determined by GC-MS and is taken as a measure of NOS activity (29)(31). The advantages of this method are its specificity for nitrite and nitrate derived from NOS activity and its potential to measure actual NO• production in vivo in humans. Disadvantages are technical complexity and costs. NOS enzyme activity is assayed by measuring the conversion of radiolabeled l-arginine into radiolabeled citrulline. This method is very sensitive and specific but requires tissue or cells. The assay can be performed on fresh material such as leukocytes and peritoneal exudate cells (32) or gastric and intestinal biopsies (9)(33)(34). The assay can also be performed on homogenates of frozen tissue or cells, provided the material is nonfixed, snap-frozen in liquid nitrogen, and stored at −80 °C. Increased NOS activity, preferably in comparison with that in control preparations, indicates induction of iNOS and inflammation. Disadvantages of this method are the requirement for tissue or cells and the necessity for handling radioactive materials. The unpaired electron of NO• can be detected by EPR spectroscopy after reaction with a spin-trap compound, i.e., a compound that reacts with radicals to form relatively stable paramagnetic species that can be detected by EPR spectroscopy (15)(18). Heme-containing proteins, particularly hemoglobin, can act as an NO• spin trap; the resulting nitrosylferroheme complexes can be detected by EPR spectroscopy (15)(18). EPR spectroscopy can be applied to erythrocytes, whole blood, or tissue specimens (15)(35). Because nitrosylferroheme complexes are relatively stable, stored material can be used for EPR spectroscopy. Recently, EPR spectroscopy with novel spin traps has been applied to detect ongoing NO• production in vivo (36)(37). EPR spectroscopy requires specialized equipment and expertise. In addition, reactions of NO• and nitrite with methemoglobin and oxyhemoglobin can interfere with the nitrosylferrohemoglobin EPR signal. Finally, near-infrared spectroscopy has been described as a technique to detect nitrosylhemoglobin formation in vivo (38). The choice for a particular technique will depend on the material available for analysis, the requested information, and the requirements for sensitivity, specificity, and speed of analysis. For routine application in a clinical chemistry laboratory, the nitrite + nitrate combination is usually measured: This determination can be applied to various body fluids, is amenable to automation, and has a high throughput. Therefore, it is important to establish the meaning and value of nitrite and nitrate determinations in body fluids. Increased nitrite + nitrate concentrations in body fluids other than blood or urine could be indicators of local inflammatory processes, as has been suggested for synovial fluid (rheumatoid arthritis) (39), bronchoalveolar lavage fluid (lung cancer) (40), sputum or saliva (inflammatory lung disorders), peritoneal fluid or ascites (peritonitis) (41), and cerebrospinal fluid (severe head injury and meningitis) (42)(43)(44)(45). Whether the increased nitrite + nitrate concentrations in cerebrospinal fluid during meningitis are related to increased production of NO• in the brain or to increased permeability of the blood–brain barrier remains to be elucidated (44)(45). Above-normal concentrations of nitrite and nitrate in these compartments presumably reflect recent or ongoing increased NO• production. However, still to be established is whether the nitrite and nitrate in a particular compartment are exclusively derived from the tissue(s) or organ(s) draining into that compartment. The most common body fluids in which nitrite and nitrate determinations are performed are plasma (serum) and urine. Among the aspects to be considered when measuring nitrite and nitrate in serum or plasma are: 1) The half-time of nitrite and nitrate persistence in blood (30). Under ordinary conditions, nitrite and nitrate are rapidly excreted into urine. This implies that increased serum concentrations of nitrite and nitrate indicate only ongoing or recently increased NO• production. 2) The effect of kidney function. Increased nitrite and nitrate concentrations do not always indicate increased production when kidney function is compromised, as was recently shown for patients with liver cirrhosis and ascites (46). Conversely, normal nitrite and nitrate concentrations do not always rule out increased production when kidney function is normal. In this regard, determining both total daily nitrite and nitrate excretion into urine as well as plasma nitrite and nitrate concentrations could be an accurate indication of total nitrite and nitrate production. 3) Sensitivity. Determination of plasma nitrite and nitrate may not be sensitive enough to detect moderate or local NO• production and inflammation, because of dilution in the large blood volume. Determination of urinary nitrite + nitrate output as a marker of inflammation has been reported for inflammatory bowel disease, allograft rejection, arthritis, gastroenteritis, and bacterial translocation (33)(47)(48)(49)(50). However, the inter- and intraindividual urinary nitrate measurements are highly variable and dependent on diet (20)(31)(51). When collecting urine for nitrite + nitrate determinations, one must take precautions to prevent bacterial growth in urine, especially in patients with urinary tract infections, because bacteria are potent producers of nitrate. Collection of urine on ice and (or) adding antibiotics or isopropanol to the urine during collection is recommended. Potentially useful applications of the determination of nitrite + nitrate in plasma or urine are (a) diagnosis and monitoring of local or systemic inflammatory processes, (b) diagnosis and monitoring of allograft rejection (52), (c) monitoring of anti-inflammatory or immunosuppressive therapy, (d) adjustment of dosage of therapeutically used NOS inhibitors, and (e) adjustment of dosage of therapeutically applied inhaled NO•, to avoid side-effects (53). Future studies need to tell us whether nitrite + nitrate determinations are really valuable in these applications. Clearly, the real value of determinations of NO• or its metabolites nitrite and nitrate in clinical practice still need to be proved. One of the major disadvantages of measuring nitrite and in particular nitrate is that these ions are naturally occurring. In addition, the range of normal values in body fluids is variable and diet-dependent. This limits the sensitivity and specificity of this assay as a marker of NO• production and inflammation and raises the question as to whether more-sensitive and specific markers of inflammation or tissue damage exist. In this respect, 3-nitrotyrosine may be a promising candidate. 3-Nitrotyrosine is formed when peroxynitrite, the result of the reaction of NO• with superoxide anions, acts on tyrosine residues in proteins (54). Simultaneous production of NO• and superoxide anions is likely during inflammation (54). Indeed, 3-nitrotyrosine residues in proteins have been demonstrated in many inflammatory disorders (6)(8)(10)(11)(55)(56)(57)(58)(59). Turnover of proteins containing 3-nitrotyrosine residues yields free 3-nitrotyrosine. Free 3-nitrotyrosine has been detected in plasma and synovial fluid of patients with rheumatoid arthritis (59). Methods to measure free 3-nitrotyrosine in body fluids and protein-bound 3-nitrotyrosine in tissue specimens after total protein hydrolysis are currently being developed (58)(59)(60). Interest in the determination of 3-nitrotyrosine as a marker of inflammation and exposure to peroxynitrite is expected to increase. In conclusion, the methodology is now operational to determine NOS, NO•, and metabolites such as nitrite, nitrate, and 3-nitrotyrosine in body fluids and tissue specimens. What needs to be established next is the value of these determinations in clinical practice.