Annals of the Rheumatic Diseases 1994; 53: 547-549 547 Ann Rheum Dis: first published as 10.1136/ard.53.8.547 on 1 August 1994. Downloaded from Increased urinary excretion in rats with adjuvant arthritis

Dirk 0 Stichtenoth, Frank-M Gutzki, Dimitrios Tsikas, Norma Selve, Stefanie M Bode-Boger, Rainer H Boger, Jurgen C Frolich

Abstract well established model ofpolyarthritis. For this Objectives-In rats with adjuvant arthritis we applied a recently developed, highly spec measurements were taken of the urinary ific and sensitive gas chromatographic method excretion ofnitrate, reflecting endogenous for determination of nitrite and nitrate in (NO) formation, and cyclic serum, urine, synovia and cell supernatants. guanosine monophosphate (cGMP). NO itself is difficult to measure directly, Methods-Urinary nitrate was deter- because of its very short half life in biological mined by gas chromatography, cGMP by fluids. NO is readily oxidised to nitrite and radioimmunoassay. nitrate,7 which are excreted rapidly into urine. Results-A significant (p < 0.001), more It has been shown, that the major source of than three fold increase of urinary nitrate urinary nitrate, in the absence of excess nitrate excretion was found in rats 20 days after intake in food, is endogenously synthesised induction of adjuvant arthritis compared NO.8 Therefore the NO synthase activity can with non-arthritic rats. There was no be assessed reliably by measuring urinary significant difference in urinary cGMP nitrate excretion, as reported by Suzuki et al9 excretion between arthritic rats and and our group (Bode-Boger et al). control animals. Conclusion-The data suggest that the dramatic increase of urinary nitrate ex- Materials and methods cretion is due to increase of NO synthesis ANIMALS AND ARTHRITIS INDUCTION by the inducible form ofNO synthase. Twelve male Sprague Dawley rats with adjuvant arthritis and 12 non-arthritic rats of (Ann Rheuni Dis 1994; 53: 547-549) the same strain and age were purchased from Charles River, Cleon, France. Adjuvant arth- http://ard.bmj.com/ ritis was induced as described by Gouret et al.'0 Nitric oxide (NO) has been shown to be As a control group 12 non-arthritic rats of the a major messenger molecule with many same strain were held under the same condi- physiological and pathophysiological func- tions. Both groups received unlimited amounts tions.' NO is synthesised from L- by of tap water (nitrate concentration <1 -6 ,umol/ the NO synthase. Two major types of this 1, nitrite concentration <0-1 ,umol/l) and food enzyme have been identified: 1) The constitu- (RM-204, Eggersmann, Rinteln, Germany). on October 1, 2021 by guest. Protected copyright. tive NO synthase is responsible for regulation of vascular tone, platelet aggregation and neuronal signal transduction; these actions are EXPERIMENTAL PROTOCOL mediated by activation of soluble guanylate At day 20 after arthritis induction secondary Department of Clinical cyclase followed by increased concentrations of arthritic lesions at all four paws, penis and tip Pharmacology, Hannover Medical cyclic guanosine monophosphate (cGMP);' 2) of the nose of each rat were examined School, The inducible NO synthase; after induction by according to the following score (maximal Hannover, endotoxin and certain cytokines, this isoform value/animal = 16): 0 = No erythema and no Germany produces large quantities of NO in contrast to inflammation; 1 = Distinct to D 0 Stichtenoth moderate ery- F-M Gutzki small amounts of NO by the constitutive form. thema of 1 paw or swelling of 1 joint at one D Tsikas The NO production by the inducible NO paw; 2 = Swelling of 2-3 joints at one paw or S M Bode-Boger synthase to R H Boger contributes the cytotoxic properties swelling ofpenis or tip of the nose; 3 = Swelling J C Fr6lich ofmacrophages,2 monocytes3 and neutrophils.4 of 4-5 joints at one paw or extensive erythema Research Centre, Although it is obvious that NO acts as part and swelling of one paw. Gruinenthal GmbH, of the cell-mediated immune response, the Twenty four hour urine samples were Zieglerstr. 6, precise role of this mediator molecule in in- collected in cages at day 20 after 52078 Aachen, Germany flammation is not clear; anti-inflammatory inoculation; at the same day the body weight N Selve properties have also been described.5 of each rat was assessed. Bacterial growth in Correspondence to: Little is known about the in vivo synthesis of the urine was prevented by addition of 2 ml Dr D 0 Stichtenoth, NO in inflammatory joint diseases. Farrell et at Department of Clinical 2-propanol into the collection tube. Pharmacology, reported elevated levels of nitrite in joint fluid Hannover Medical School, and serum of patients with rheumatoid 30623 Hannover, Germany. arthritis. We have therefore studied the ANALYSES Accepted for publication excretion of the major urinary metabolite of Urinary nitrate was determined by a gas 11 May 1994 NO, nitrate, in rats with adjuvant arthritis, a chromatographic method, based on a reaction 548 Stichtenoth, Gutzki, Tsikas, et al

of nitrate with trimethoxybenzene to form trimethoxynitrobenzene. Fifty pI aliquots of urine were diluted with 50 RI double-distilled Ann Rheum Dis: first published as 10.1136/ard.53.8.547 on 1 August 1994. Downloaded from water and treated with 300 pLI silver sulphate solution (100 mg/ml) for chloride precipita- tion. After centrifugation (5 min, 8000 X g) 300 pI aliquots of the supernatant were mixed with 300 plI of concentrated sulphuric acid, 20 pI trimethoxybenzene in acetone (1 mg/ml) and 10 [lI dimethoxynitrobenzene in acetone (250 ng/fl) as internal standard. After incubation (10 minutes at 60°C) the generated nitroaromates were extracted with 800 pI of toluene. The toluene layer was drawn off and shaken with 2 ml of a 4 M aqueous solution. Subsequently 100 pAl ofthe toluene phase were diluted with 900 pI of toluene for gas chromatographic analysis. Gas chromatography was performed on a Carlo Urinary nitrate excretion in arthritic and non-arthritic Erba HRGC5 160 (Fisons Instruments, Mainz, control rats. Mean (SE), n = 12. Germany) equipped with an A200S auto- **p < 0 001 versus controls in two-tailed, unpaired t test. sampler and an ECD HT40 electron capture detector. An OV1701 fused silica capillary rats [510 (44) nmol/mmol creatinine] than in column (Machery and Nagel, Diiren, controls [747 (33) nmol/mmol creatinine]. Germany) was used for chromatographic sep- There was no difference in urinary creatinine aration. Helium (75 kPa) was applied as carrier excretion between arthritic [72 (5) pumol/d] gas and (150 kPa) as make up gas. and non-arthritic rats [68 (3) limol/d]. Oven temperature was held at 150°C for two minutes then programmed to 280°C at a rate of 40°C/minutes and held at 280°C for two Discussion minutes. The intra- and interassay coefficients In the present study we have demonstrated that of variation were below 3 5%. The detection the urinary nitrate excretion was increased limit of the method was 5-2 nmol nitrate/ml. more than three fold in rats suffering from The method was validated by a recently adjuvant arthritis compared with healthy published gas chromatography/tandem mass controls. There was no higher food intake in spectrometry assay" and showed a coefficient arthritic rats, resulting in higher body weight, of correlation of r = 0 91, n = 15, in the range which could account for this result; on the of urinary nitrate levels. contrary, the arthritic animals had lower body http://ard.bmj.com/ For the determination of cGMP levels in weights than the non-arthritic rats. The urine we used a radioimmunoassay method as drinking water did not measurably contribute described previously.'2 to urinary nitrate excretion. Therefore higher Urinary creatinine was determined spectro- urinary nitrate excretion due to excess food photometrically using the alkaline picric acid and water intake can be ruled out and the method in an automatic analyser (Beckman, elevated urinary nitrate excretion of arthritic Galway, Ireland). The urinary excretion rates rats suggests elevated NO synthesis. on October 1, 2021 by guest. Protected copyright. of nitrate and cGMP were corrected by Ialenti et al'3 demonstrated elevated nitrite creatinine excretion. generation by peritoneal macrophages col- lected from rats with adjuvant arthritis com- pared with controls; nitrite generation and the STATISTICS severity of arthritis was exacerbated by Data are expressed as mean (SD). Statistical L-arginine, the source for NO production, and significance of differences was determined by supressed by N0-nitro-L-arginine methyl , an unpaired t test. A p-value <0 05 was an inhibitor of NO synthesis. considered significant. In rats with arthritis, induced by injection of streptococcal cell wall fragments, McCartney- Francis et all4 found elevated NO production Results by synovial tissue of inflamed joints and The mean (SD) arthritis score was 10 (1) in by polymorphonuclear cells. Administration arthritic animals at day 20 after induction of of NG-monomethyl-L-arginine profoundly adjuvant arthritis. In the non-arthritic rats no reduced NO production by synovial tissue, signs of inflammation could be examined, synovial inflammation and tissue damage. In therefore the arthritis score of each rat was 0. our study urinary cGMP excretion was not The body weight was significantly (p < 0-001) increased in rats with adjuvant arthritis in spite lower in arthritic rats than in healthy control of a more than three fold increase in urinary animals [218 (5) g versus 242 (4) g]. nitrate excretion. This points to an activation The urinary nitrate excretion was sig- of the inducible form of the NO synthase, nificantly increased in rats suffering from arth- because urinary nitrate and cGMP excretion ritis compared with the non-arthritic control are influenced in parallel when the constitutive group (figure). Urinary cGMP excretion was NO synthase is activated, as reported by slightly, but not significantly lower in arthritic Kanno et al.'5 Increased urinary nitrate excretioni i.n rats with adjuvant arthritis 549

In summary, endogenous NO formation by 6 Farrell A J, Blake D R, Palmer R M J, Moncada S. Increased concentrations of nitrite in synovial fluid and serum the inducible NO synthase is enhanced in samples suggest increased nitric oxide synthesis in

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