NITRITE LEVEL OF SERUM AS A DIAGNOSTIC AND PROGNOSTIC TOOL IN PATIENTS WITH RHEUMATOID ARTHRITIS

G.R. Moshtaghi Kashanian PhD

Hospital No. 1, Kerman University of Medical Sciences, Kerman, Iran

Introduction

Nitric oxide (NO) is the first well-characterised representative of a class of gaseous biological mediators,^1-3 which may also include oxygen intermediates⁴ and carbon monoxide.^5,6 NO is produced by the oxidation of the guanidine nitrogen of arginine by molecular oxygen. The enzyme responsible being named NO synthase (NOs), and at least two main classes of different NO synthases have been identified, three of which have been cloned in various cells including neuronal and endothelial cells, and macrophages.^2,3,7

The first class of NO synthases are constitutively active and regulated by calcium and calmodulin.^3,7-9 This class includes neuronal and endothelial NOs (cNOs), that produce pico- to nano-mole of NO for short periods, in response to receptor stimulation such as acethylcholine or shear stress.¹⁰ NO produced by the endothelial cells is the major component of endothelium-derived relaxing factor, which regulates vasodilator tone,¹¹ while NO produced by neurons throughout the brain, acts as a neurotransmitter.^7,12 This NO is involved in induction of growth hormone secretion from pituitary cells by GH-releasing hormone.¹³ It is also produced in nonadernergic, noncholinergic peripheral nerve cells,¹⁴ which, are probably induced by vasoactive intestinal peptide (VIP), as is the case in the gastric muscle cells,¹⁵ rat pinealocytes¹⁶ and many other tissues such as smooth muscle,¹⁷ intestinal, sphincteric visceral, corpora cavernosa, and cerebral vessels.¹⁵

The second class which is referred to as inducible (iNOs) contains calmodulin as an integral component and is not affected by external calcium concentration.¹⁸ This enzyme is a cytosolic enzyme of many cells, such as macrophages, endothelial cells, chondrocytes, hepatocytes, synoviocytes and smooth muscle cells.¹⁰ Inducible NOs is induced by inflammatory stimuli,^19-21 and NO produced by iNOs is a vital component of the tumouricidal and fungicidal apparatus of macrophages.^22-24 The expression of NOs is regulated by the balance of cytokines in the micro-environment; for example, transforming growth factor ß, IL-10 inhibit iNOs expression in macrophage.¹⁰

NO being a gaseous free radical, has a half life of less than 15 second^2,3,10 and is rapidly metabolized to nitrate.^25,26 Although nitrate and nitrite are the most common end products of NO, there are many other reactions such as formation of complexes with transitional metal ions, and free thiols groups,²⁶ in glutatione and albumin that have NO-like properties with extended half life of more than 2 hours.^27-30

There is evidence, that increased amounts of blood and urine nitrate, can be detected in vivo during infections³¹ following cytokine administration.³² Increased levels of nitrate has been detected in many disease such as sepsis,^33,34 ulcerative colitis,³⁵ arthritis³⁶ multiple sclerosis,³⁷ and type I diabetes.³⁸ Excessive NO is also produced during the course of a variety of rheumatic disease, including systemic lupus erythematosus, Sjogren’s syndrome, vasculitis, osteoarthritis, and rheumatoid arthritis.³⁹

Since it is difficult to measure NO directly because of its short half-life, nitrate (NO₃-) or nitrite (NO₂-) are measured normally as indices of NO production. The blood concentration of nitrite is much lower than that of nitrate, but there is far more nitrate in the diet,⁴⁰ so measurement of nitrite avoids the problem of variation caused by dietary intake.

Previously, we developed a method of measurement of nitrite and nitrate in serum and other biological fluids.⁴¹ In the present study, we have adapted these methods in patients with rheumatoid arthritis.

Material and Methods

All necessary chemicals were provided by Sigma, except cadmium granules (5-20 mesh) which were purchased from Aldrich (Dorset, UK). Centriflo membrane filter cones (CF-25) which were used for deproteinization of serum were bought from Amicon (Stonehouse, Glos, UK).

Blood (6-8 ml) collected from 40 healthy adults aged 25-70 years (F/M=2/1, 52.60 ± 12.83) served as the control group, while blood samples collected from 64 patients with known rheumatoid arthritis (F/M=3/1), who attended the out patient department of the Rheumatoid Clinic of Gartnavel General Hospital (Glasgow. UK) served as our study group. These patients were aged between 26 to 90 years (63.53 ± 13.32). The samples were collected in plain tubes, and serum was separated within one hour of analysis. If samples were not analysed on the days of collection, sera were kept at –20 °C until the day of analysis.

For measurement of nitrite and nitrate, methods developed by our group⁴¹ based on Griess reaction⁴² were used. It is necessary to use protein free samples, so for nitrite assay, samples were deproteinized by centrifugation using Centriflo membrane cones type CF-25. This method gives deproteinized serum without dilution. The membranes were prepared for use according to the manufacturer’s instructions. Two ml of serum were pipetted into each cone and centrifuged at 920 g for 35 minutes. Filtrates were used for nitrite assay. For measurement of nitrate, samples were deproteinized using Somogyi’s method,⁴³ with a dilution factor of 1 to 4.

In nitrite assay, to one ml of distilled water, filtrate or working standards, 105 µl of sulphanilamide solution (58 mmole/l in 3 molar HCl) and Naphthylethylenediamin solution of 772 µmole/l) were added. Tubes were mixed and after 15 minutes, absorbance was measured at 543 nm, against distilled water as blank.

For measurement of nitrate, copper coated cadmium granules were used to convert nitrate to nitrite. For this step, cadmium granules that were stored in 0.1 molar sulphuric acid, were washed by swirling them with distilled water. Then a solution of copper sulphate (15 mmole/l in 0.2 mole/l glycine buffer, pH 9.7) was used to coat the granules, by swiming them for two minutes. Cadmium granules were drained and dried over tissue paper, and used within five minutes.

To reduce nitrate to nitrite, 0.5 ml of distilled water, standards, or deproteinized samples were added to labelled tubes, followed by 0.5 ml of glycine buffer (0.2 mole/l, pH=9.7), and 2-3 grams of copper coated cadmium granules were added.

The tubes were shaken for 15 minutes on an electric shaker. After the reduction step, 0.5 ml of the sample or standard was transferred to an appropriate labelled tube, followed by the addition of 0.5 ml of freshly prepared colour reagent. Tubes were incubated at room temperature for colour development (15 minutes) and then absorbance was measured at 543 nm, using distilled water tubes as blank. C-reactive protein (CRP) was measured for all patients and control groups, using protocol provided by the kit manufacturer (BioMérieuv).

Statistical analysis was carried out using Stat View package. When the numbers of data were equal, paired t-test was used, while unpaired t-test was carried out for comparisons of sets of unequal data. Simple Pearson’s regression procedure was used when correlation of independent parameters was compared.

Results

Determined CRP reference value for control group was 4.63 ± 2.18 mg/l (mean ± std deviation). This value for patients group ranged 8-159 mg/l (38.8 ± 34.5). Among patients groups, there were 17 patients with CRP levels of less than or equal to 10, these patients were classified as inactive cases. Five patients with CRP levels of 11 and 20 were classified as borderline cases.

The 26 patients with CRP levels of 21-50 were classified as stage one, 11 patients with CRP of 51-100 as stage 2, and 5 patients with CRP levels of more than 100 as stage 3. The reference value obtained for serum nitrite levels from the control group was 262 ± 34 nmole/l. This value was 329 ± 55 nmole/l for the inactive cases. Although there was a small increase in the level of nitrite of this group, statistically the difference was not signi-ficant when compared with corresponding values obtained for reference groups.

The level of nitrite increased significantly (p<0.01) in the serum of patients whose CRP levels were in the range of 11 to 20 (470 ± 37 nmole/l) or 20 to 50 mg/l (488 ± 11.7 nmole/l). There was no significant difference between the values obtained for these groups, this indicates that these patients could be at the same stage of disease, and should be classified as one group with mild but active rheumatoid arthritis. The mean value obtained for the nitrite of patients whose CRP was between 51 and 100 was 705 ± 10.1 nmole/l. This increase is highly significant when compared with the value obtained for the reference group (p<0.0001). It was also significantly different from the value obtained for first, second, and third groups of patients (p<0.001). The mean value obtained for the nitrite level of patients with CRP levels of over 100 was 1.03 ± 0.089 µmole/l. This value was significantly higher (p=<0.0001) than that obtained for other group of patients and the reference group (Fig. 1).

The mean value obtained for nitrite level of the reference group was 37.5 ± 7.60 µmole/l, while the values for patient’s group were 42.82 ± 14.99 µmol/l. The only group with a statistically significant increase in the level of nitrate was the last group of patients with CRP levels of more than 100 mg/l that had nitrate levels of 58.82 ± 14.99 µmole/l (p<0.05).

To determine the correlation between nitrite or nitrate and CRP, simple regression analysis was carried out for all patients. The regression analysis for nitrite and CRP showed significant correlation between them (p<0.0001, and R=0.892), while corresponding values for nitrate indicated a lower correlation (p=0.0229, and R=0.273). The regression curves are shown in Fig. 2.

Discussion

Rheumatoid arthritis is an autoimmune disease, with unknown auto-antigen.⁴⁴ Since the target of the disease is the joints, the collagen present at these sites is one of the probabable auto-antigens.⁴⁴ The inflammation of joints is one of the symptoms of the disease, which is characterized by marked mononulcear (macrophages, plasma cells, and lymphocytes) infiltration and microvascular proliferation.³⁹ Induction of iNOs of the infiltrated cells, synovial lining layer, cartilage, chondrocytes, and fibroblasts by pro-inflammatory cytokines (IL-1,IL-6, TNF-a, and TGF-b), release large amounts of NO, that leads to local (synovial fluid) and systemic increases of nitric and nitrate.^45,46

In the present study, the serum concentrations of nitrite and nitrate of patients with rheumatoid arthritis were analysed, without considering the course of treatments or the stage of the disease. All patients had higher levels of nitrite compared to the control group, but only patients in stage 3 (CRP>100 mg/l) had high level of nitrate. This result confirms the in vitro^45-46 and in vivo reports⁴⁷ that showed increases in nitrite and nitrate levels of the synovial fluid, urine or serum of the patients with active rheumatoid arthritis. In addition, the patients in our study were undergoing different courses of treatment, but were not on a restricted diet. Since only patients with high levels of CRP did have high levels of nitrate, it may be concluded that except in active cases of the disease release of NO is high (nitrate or nitrite levels are high), dietary nitrate could conflict with nitrate produced in vivo, and measurement of nitrate could be deceptive.⁴⁰ Finally, it can be concluded that nitrite levels of serum is a better marker of rheumatoid arthritis than nitrate levels of serum as previously suggested.⁴⁸

Usually, the activity of the disease is categorized according to CRP levels or erythrocyte sedimentation rate (ESR). In this study, patients were categorized according to their CRP levels. Simple regression analysis showed that patients with higher CRP levels had higher nitrite concentration, and there is a highly significant correlation between nitrite and CRP levels (R=0.892, p<0.0001). This result may provide indirect evidence of induction of iNOs as well as hepatic synthesis and release of CRP by pro-inflammatory cytokines.^10,46 Furthermore, it may show that these cytokines are also elavated in the circulating system that may induce the production and release of CRP by hepatocytes.

There are many cells that synthesis NO through induction of one or two classes of NO synthesis.

Though normal levels of NO are essential for living, high concentrations of NO can be cytotoxic.¹⁰ Among the patients with rheumatoid arthritis, the cells that can contribute part of elevated NO (nitrite) in the site of inflammation are fibroblast, cartilage, chondrocytes, and synovial lining layer, due to the release of cytokine at the site of inflammation,^45,46 while NO synthesised by iNOs of endothelial cells, infiltrated cells and immune-cells in extra cellular fluid could be another source of elevated nitrite, due to the elevation of circulation pro-inflammatory cytokines.⁴⁵ NO released locally changes the normal function of osteoblast and osteoblast cells¹⁰ and could therefore be the cause of pathogenesis of bone loss, and pain.⁴⁹ The present results, show that the measurement of nitrite that is a direct index of NO production, could be a diagnostic, as well as prognostic, tool especially during treatment of patients with NOs inhibitors.

References

1. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB Journal 1992;6:3051-64.
2. Moncada S. The L-arginine nitric oxide pathway. Acta Physiol Scand 1992;145:201-27.
3. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. New Eng J Med 1993;329:2002-12.
4. Schreck R, Baeuerle PA. A role for oxygen radicals as second messengers. Tren Cell Biol 1991;1:39-42.
5. Verma ADJ.Carbon monoxide: A putative neural messenger. Science 1993;259:381-4.
6. Pozzoli G, Mancuso C, Mirtella A, et al. Carbon monoxide as a novel neuroendocrine modulator: Inhibition of stimulated corticotropin-releasing hormone release from acute rat hypothalamic explants. Endocrinology 1994;135:2314-7.
7. Bredt DS, Snyder SH. Nitric oxide: A physiologic messenger molecule. Ann Rev Bioch 1994;63:175-95.
8. Bredt DS, Snyder SH. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990;87:682-5.
9. Bredt DS, Hwange PM, Glatt CE, et al. Cloned and expressed nitric oxide synthase structurally resembles cytochrome p-450 reductase. Nature (London) 1991;351:714-8.
10. Clancy MC, Amin AR, Abramson SB. The role of nitric oxide in inflammation and immunity. Arthritis Rheum 1998;41:1141-51.
11. Palmer RMJ, Ferrige AJ, Moncada S. Nitric oxide release account for the biological activity of endothelium-derived relaxing factor. Nature (London) 1987;327:524-9.
12. Garthwaite J, Charles S L, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature (London) 1988;336:385-8.
13. Kato M. Involvement of nitric oxide in growth hormone (GH) -releaseing hormone-induced GH secretion in rat pituitary cells. Endocrinology 1992;131:2133-8.
14. Li CG, Rand MJ. Evidence for a role of nitric oxide in the neurotransmitter system mediating relaxation of rat anococcygeus muscle. Clin Exp Pharmacol Physiol 1989;16:933-8.
15. Murthy KS, Zhang KM, Jin JG, et al. VIP-mediated G protein-coupled Ca²⁺ influx-activates a constitutive NOs in disperesed gastric muscle cells. Am J Physiol 1993; 265:G660-G671.
16. Spessert R. Vasoactive intestinal peptide stimulation of cyclic guanosine monophosphate formation: Further evidence for a role of nitric oxide synthase and cytosolic guanylate cyclase in rat pinealocytes. Endocrinology 1993;132:2513-7.
17. Grider JR, Murthy KS, Jin JG, Makhlouf GM. Stimulation of nitric oxide from muscle cells by VIP: prejunctional enhancement of VIP release. Am J Physiol 1992;262:G774-G778.
18. Cho HJ, Xie QW, Calaycay J, et al. Calmodulin as a tightly bound subunit of calcium, calmodulin-independent nitric oxide synthase. J Exp Med 1992;176:599-604.
19. Ding AH, Nathan CF, Stuehr DJ. Release of active nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for indepent production. J Immunol 1988;141:2407-12.
20. Drapier JC, Wietzerbin J, Hibbs JB Jr. Interferon-? and tumor necrosis factor induce the L-arginine-dependent cytotoxic effect mechanism in murine macrophages. Eur J Immunol 1988;18:1587-92.
21. Hunt NCA, Goldin RD. Nitric oxide production by monocytes in a alcoholic liver disease. J Hepatol 1992; 14:146-50.
22. Hibbs JB Jr, Taintor RR, Vavrin Z. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 1987;235:573-6.
23. Stuehr DJ, Nathan CF. nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989;169:1543-5.
24. Granger DL, Hibbs JB Jr, Perfect JR, et al: Metabolic fate of L-arginine in relation to microbiostatic capability of murine macrophages. J Clin Invest 1990;85:264-73.
25. Marletta MA. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 1994; 78:923-30.
26. Stamler j, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redoxactivated forms. Science 1992; 285:1898-902.
27. Clancy RM, Abramson SB.Novel synthesis of S-nitrosoglutathione and degardation by human neutrophils. Anal Biochem 1992;204:365-71.
28. Myers PR, Minor RL Jr, Guerra R Jr, et al. Vasorelaxant properties of endothelium-derived relaxing factor more closely resemble S-nitrosothiol than nitric oxide. Nature 1990; 345:161-3.
29. Stamler JS, Jaraki O, Osborne J, et al: Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci USA 1992;89:7674-7.
30. Clancy RM, Levartovsky D, Leszzczynska-Piziak J, et al. Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: Evidence for S-nitrosoglutathione as bioactive intermediatory. Proc Natl Acad Sci USA 1994;91:3680-4.
31. Ochoa JB, Udekwu AO, Billiar TR, et al. Nitrogen oxide level in patients after trauma and sepsis. Ann Surg 1991;214:621-6.
32. Hibbs JB Jr, Westenfelder C, Tainter R, et al. Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy. J Clin Invest 1992;89: 867-77.
33. Hutcheson AR, Whittle BJR, Boughton-Smith NK. Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat. Br J Pharmacol 1990;1282:7889-91.
34. Petros A, Bennett D, Vallance P. Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet 1991;338: 1557-8.
35. Middleton SJ, Shorthouse M, Hunter JO. Increased nitric oxide synthesis in ulcerative colitis. Lancet 1993;341:465-6.
36. McCartney-Francis N, Allen JB, Mizel DE, et al. Suppression of arthritis by an inhibitor of nitric oxide synthesae. J Exp Med 1993;178: 749-54.
37. Parkinson JF, Mitrovic B, Merrill JE: The role of nitric oxide in multiple sclerosis. J Mol Med 1997;75:174-86.
38. Klob H, Kolb-Bachofen V. Type I (insulin-dependent) diabetes mellitus and nitric oxide. Diabetologia 1992;35:796-7.
39. Clancy RM, Abramson SB. Nitric oxide: a novel mediator of inflammation. Soc Exp Biol Med 1995;210:93-101.
40. Knight TM, Forman D, Al-dabbagh SA, Doll R. Estimation of dietary intake of nitrate and nitrite in Great Birtain. Food Chem Toxicol 1987;25:277-85.
41. Moshtaghi Kashanian GR, Shapiro D. Rapid methods for analysis of nitrite and nitrate in serum of other biological fluids: Second Conference of Graduate Research on Medical Sciences Leuven; Belgium: April 1994.
42. Nicholas DJD, Nason A. Determination of nitrate and nitrite. Methods Enzymology 1957;3:982-4.
43. Somogyi M. A method for the preparation of blood filtrates for the determination of sugar. J Biolog Chem 1930;86:655-63.
44. Janeway CA Jr., Travers P. Immunobiology: The immune systems in health and disease. Garland publishing Inc. London. 1994.
45. Ralston SH: Nitric oxide and bone: what a gas! Br J Rheumatol 1997;36:831-8.
46. Betero MT, Caligaris-Cappio F. Anemia of chronic disorders in systemic autoimmune disease. Haematologica 1997;82:375-81.
47. Wigand R, Meyer J, Busse R, Hecher M. Increased serum NG-hydroxy-L-arginine in patients with rheumatoid arthritis and systemic lupus erythematous as an index of an increased nitric oxide synthase activity. Ann Rheum Dis 1997;56:330-2.
48. Grabowski PS, England AJ, Dykuizen R, et al. Eleveated nitric oxide production in rheumatoid arthritis. Detection using the fasting urinary nitrate: creatinine ratio. Arthritis Rheum 1996;39:643-7.

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