An Endogenous Source of Elevated Nitrite in Infected Urine

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Kidney International, Vol. 45 (1994), pp. 586—591

Nitric oxide synthase: An endogenous source of elevated nitrite in infected urine

SHANNON D. SMITH, MARCIA A. WHEELER, and ROBERT M. WEIss

Section of Urology, Yale University School of Medicine, New Haven, Connecticut, USA

Nitric oxide synthase: An endogenous source of elevated nitrite inwe measured both the nitrite concentration and nitric oxide infected urine. To further define the endogenous sources of urine nitrite in urinary tract infections, we measured urinary nitrite levels by the synthase activity in human infected and non-infected urine. Griess method and assayed urinary nitric oxide (NO) synthase activity by the conversion of '4C-arginine to '4C-citrulline. Endogenous produc- Methods tion of '4C-citrulline was confirmed by thin layer chromatography. Exogenous L-arginine increased nitrite production in whole infected Urine sample isolation urine, but not in bacteria isolated from infected urine. Urinary tract Urine specimens from symptomatic emergency room and infections significantly increased NO synthase activity in soluble urine fractions, although soluble activity was less than 10% of particulate urology clinic patients (N =18)were submitted to the Clinical activity. Urine particulate fractions from women with non-infectedMicrobiology Laboratory at the Yale-New Haven Hospital urine had greater NO synthase activity than particulate fractions from after routine urinalysis. The majority (87%) were clean-catch men with non-infected urine, 11 2and 0.2 0.1picomol/minlmg midstream specimens; the remainder (13%) were obtained by protein, respectively. Urinary tract infections increased NO synthase sterile catheterization. Patients' symptoms included dysuria, activity in urine particulate fractions from women and men, 99 20and 48 9picomollmin/mg protein, respectively. The conversion of '4C- frequency, urgency, lower abdominal and flank pain, and fever. arginine to '4C-citrulline required NADPH, was calcium independent, For the purpose of this study, significant bacteriuria was and was inhibited to a greater extent by L-canavanine than by N°-defined as a colony count of greater than io CFU/ml of a monomethyl-L-arginine or N°-nitro-L-arginine. Human infected urine clearly predominating microorganism. Escherichia coli (E. coli) contains an isoform of NO synthase which is an endogenous source of urine nitrite. grew in sixteen samples, Serratia in one sample, and Kiebsiella in one sample. Ten control urine specimens were obtained from asymptomatic men and women. Control urine samples were negative by reagent strip urinalysis and had less than five white A direct relationship exists between urine nitrite levels andblood cells per milliliter by microscopic exam. All control urine urinary tract infection [1]. The nitrite test, first described byspecimens were clean-catch midstream specimens. Griess [21, has been used as a simple and rapid method for detecting bacteriuria since many bacterial species convert en- Filtration of urine dogenous nitrate to nitrite. Nitric oxide (NO) is a potential To isolate bacteria from inflammatory cells, urine (3 ml) was endogenous source of infected urine nitrite detected by thefiltered through a sterile, non-pyrogenic nylon filter with a 5.0 Griess reagent. NO synthase, which has been localized inm pore size (Micron Separations Inc., Westboro, Massachu- several mammalian cells including macrophages, convertssetts, USA). Bacteria were, thus, isolated in the filtrate as L-arginine to nitric oxide and citrulline in a NADPH-dependentconfirmed by microscopy. Leukocytes did not pass through the reaction [3—5]. Nitrite is an oxidation product of nitric oxide. Infilter. Because bacteria may contaminate the leukocyte frac- rodents, the induced form of NO synthase has been defined astion, the filtrates (bacteria-enriched samples) were used in the a source of reactive nitrogen intermediates in anti-tumor andGriess-nitrite determinations. anti-microbial activities; however, defining human macrophage NO synthase activity has been difficult [6—8]. Human macro- Griess reaction phages derived from cultured peripheral blood monocytes have not consistently produced detectable nitrite levels in response The nitrite concentration in whole urine and urine filtrate was to stimulus [7]. Therefore, to elucidate the mechanisms ofdetermined by the Griess reaction [2]. Nitrite reacts with 1% urinary nitrite formation detected by the Griess reaction and tosulfanilamide in 5% H3P04/0. 1% naphthalene-efhylenediamine characterize human inflammatory nitric oxide synthase activity,dihydrochloride forming a chromophore (pink to dark red color) absorbing at 546 nm. The Griess reaction was performed immediately after receiving urine samples and was repeated after 37°C incubation of urine for four and eight hours. The Received for publication June 10, 1993 Griess reagent (500 l) was added to urine (100 /hl) and H20 (400 and in revised form August 25, 1993 pi) or NaNO2 standards (0.1 to 4.0 jsg/500 .d H20) and the Accepted for publication August 26, 1993 samples and standards were incubated 15 minutes at room © 1994 by the International Society of Nephrology temperature before reading the absorbance.

586 Smith et a!: NO synthase and nitrite in urinary infection 587

Assay of nitric oxide synthase 5 Urine (stored on ice) was centrifuged at 1700 x g (10 mm, 0°C) to isolate the pellet [9]. The urine pellet was resuspended 4 in ice-cold phosphate buffered saline and centrifuged at 1700 x g. The resultant pellet was resuspended in an ice-cold homog- * enization buffer (1 vol pellet/b vol buffer) which consisted of 20 3 mM Hepes (pH 7.2), 1.0 mrvi DTF, leupeptin (0.2 mg/lOO ml) and 1.0 mg/100 ml of soybean trypsin inhibitor, pepstatin, PMSF, 2 antipain, and chymostatin. The resuspended pellet was homog- enized (10 seconds, at 70% power) using a Polytron PT 10/35 homogenizer equipped with a PTA 7 K! generator (Brinkmann, Westbury, New York, USA) and the homogenate was centrifuged at 27,000 x g for 45 minutes at 4°C. The resultant particulate fraction was resuspended in 1.5 ml of homogeniza- 0 tion buffer containing 1.0 M KCI after isolation of supernatant. The particulate fraction was centrifuged at 27,000 X g for 45 I I - minutes (0°C), resuspended in homogenization buffer, centri- Time 0 4 hours 8 hours fliged 27,000 x g for 45 mm (0°C), resuspended in homogeni- Fig.1.Effectof infection and incubation time on urine nitrite produc- zation buffer, centrifuged 27,000 x g for 45 minutes andtion as measured by the Griess method. Each data point is the mean resuspended again in homogenization buffer so that the finalSEM of the urine nitrite concentrations from at least five different enzyme hydrolyzed less than 30% of '4C-L-arginine. The su-donors. Asterisk represents a 95% significance level between control pernatant was passed over a 1.0 ml AG 50W-X8 column to(A) and infected (0) urine. remove endogenous arginine before assay. Samples were stored at —80°C until assay without loss of activity. Nitric oxide synthase activity was measured as the formationPost-column-separation samples (50 to 100 d) and 10 p1 of of '4C-citrulline from '4C-arginine. The particulate or solubleL-arginine (1.0 mg/mI), ornithine (1.0 mg/ml) and citrulline (1.0 enzyme (150 d) or buffer control (150 p1 of homogenizationmg/ml) were applied to silica-gel plates (Sigma, #T-6395) and buffer) and 50 p1 of homogenization buffer containing (finaldeveloped with a chloroform-methanol-ammonium hydroxide- concentration): 3 sM '4C-L-arginine (0.2 .tCi), 2.0 mMwater solvent system (0.5 : 4.5 : 2.0: 1.0). NADPH, and 1.0 mri CaC12 were incubated at 37°C for 45 minutes. The reaction was linear for at least 45 minutes. NO Materials used synthase inhibitors, when used, were added prior to incubation. NGmonomethylLarginmne, NGnitroLarginine, L-canava- The assay was terminated with 1.0 ml of 20 ifiM Hepes (pH 5.5)nine, NADPH, and all protease inhibitors were obtained from containing 2.0 mri EDTA. The sample was applied to a 1.0 mlSigma Chemical Company (St. Louis, Missouri, USA). column of AG 50W-X8 (Na form) and eluted with 1.0 ml ofL-[14C(U)]-arginine (340 mCi/mmol) was obtained from Du- Hepes (pH 5.5). The column effluent was measured in a liquidPont/NEN (Wilmington, Delaware, USA). AG 50W-X8 (Nat scintillation counter. The buffer control cpm values were sub-form) was obtained from Bio-Rad (Richmond, California, tracted from all other sample values. Protein concentrationsUSA). were measured by the Bio-Rad Protein Assay using bovine gamma-globulin as a standard. Insoluble samples were hydro- Data and statistical analyses lyzed in NaOH prior to the protein assay. The average protein Results were expressed as mean SEM. Means were com- in supernatant and particulate fractions was 0.23 0.03 mg/mlpared using analysis of variance (ANOVA) followed by the and 0.24 0.05 mglml, respectively. Scheffe F-test. Multi-comparison significance level was 95%.

Determination of Km Results The Km values were determined by computer-assisted curve Urine and urine-filtrate nitrite levels in the presence of fitting to a predefined Michaelis-Menton equation. Four partic- arginine or nitrate ulate urine samples were passed over a 1.0 ml column of AG Nitrite levels in control and infected urine samples were SOW-X8 (Nat form) to remove endogenous arginine and then,measured by the Griess method immediately after receiving arginine (3 /sM to 200 M) was added and the 14C-citrullineurine samples and after urine incubation (37°C) for four and production at these concentrations fitted on the curve. eight hours. Samples were also exposed to 1.0 mri L-arginine or 1.0 mM NaNO3 (nitrate) and incubated for four hours. Control Thin layer chromatography urine samples, after addition of the Griess reagent, did not After completing the enzyme assay and column separation,change color. Control urine nitrite levels, measured by spectro- thin layer chromatography [10] was performed on control bufferphotometry, were always less than infected urine levels and did samples and on samples containing particulate fractions fromnot change with time or the addition of L-arginine or nitrate. non-infected urine, infected urine, urine filtrate, or E. coli Figure 1 shows the significant elevation of nitrite with time in (isolated by the Yale-New Haven microbiology laboratory frominfected urine samples. The nitrite concentration measured in urine cultures grown on blood and MacConkey agar plates).infected urine after incubation (37°C, 4 hr) was compared to 588 Smith et a!: NO synthase and nitrite in urinary infection

200 * A Particulate fraction B Soluble fraction 150 10 175 0. 0. a) 0> 150 8 0 100 125 6 0C 0100 a) > ci) 4 0 75 50 E a> (I) 0 Co 2 a) 50 0 C 25 0 0 I Male Female 0 Male Female 0 Fig. 3. A and B. DUferences in urine particulate and soluble '4C- product activity between men and women with urinary tract infections —25 (B) and without urinary tract infections (U,control).Each value, Whole urine Urine Filtrate expressed as pmol '4C-product/min/mg protein, is based on assay determinations from at least 5 different patients. Significance between Fig. 2. Percent change in nitrite concentration after addition of argi- any of the four data points for particulate '4C-effluent activity is greater nine () or nitrate (U) to infected urine samples before and afterthan 95%. Significance between control and infected soluble '4C- filtration through a 5 iun pore filter. The nitrite concentration waseffluent activity is greater than 95%. measured in infected urine (N =II)and urine filtrate (N =3)after incubation (37°C, 4 hr) and compared to incubated infected urine exposed to 1.0 mai arginine or nitrate. The percent increase in nitrite concentration is reported as the mean SEM. Asterisk represents P < Table 1. TLC of column effluent samples after NO synthase assay 0.01. Effluent Citrulline Citrulline TLC sample cpm/100 ,td % cpm Buffer Control 458 37 169 values obtained from infected urine exposed to 1.0 mM L-argi-E. coli 3366 13 438 nine or 1.0 mM nitrate and incubated for four hours at 3TC. Filtrate (infected urine) 1900 5 95 Infected urine nitrite production increased 102 27% with the Control male urine 330 28 92 addition of exogenous arginine and 124 35% with the additionControl female urine 7950 75 5963 of nitrate (N = Infected male urine 8690 97 8429 11).In urine filtrate samples, exogenous arginineInfected female urine 16460 82 13497 did not increase nitrite production; however, nitrate did (33 After completing the enzyme assay and column separation, TLC was 11%). All percent increases were significant (P < 0.01, N =3;performed on the above named effluent samples. "Buffer control" Fig. 2). contains the Hepes assay solution but does not contain bacteria or urine. The effluent radioactivity was measured prior to silica-gel-plate NO synihase activity in urine pellet isolated from men and application (cpm/100 1u1 effluent). The area on the plate corresponding to women with and without genitourinary tract infection each sample's citrulline production (R =0.9)was isolated, counted, and reported as a percentage of effluent radioactivity. As shown in Figure 3A, >4C-product activity (pmol arginine metabolized/mm/mg protein) was significantly elevated in male and in female infected urine pellets (49 9 and 121 25, respectively). Particulate 14C-product activity in women without urinary tract infection was 18 times greater than in menCitrulline was clearly separated from ornithine and arginine by without infection (14 3 and 0.8 0.2, respectively). 14C-TLC; the Rf values for arginine, ornithine, and citrulline were product activity in the supernatant, while much lower than the0.52, 0.77, and 0.92, respectively [10]. NO synthase activity particulate effluent activity increased four- and 11-fold in menwas measured in samples containing only homogenization and women with infection when compared to uninfected con-buffer and in the particulate fractions from control urine, trols (Fig. 3B). The Km for enzyme activity, using 3.0 to 200 tMinfected urine, infected urine filtrate, or E. coil. As shown in arginine substrate, in urine particulate fractions was 18 5 MTable 1, there was no evidence of NO synthase activity in urine L-arginine (N =4). filtrate samples. Radioactivity that migrated like citrulline was a smaller percentage of the total radioactivity in E. coil, urine Thin layer chromatography of control urine (without ifitrate, and male control urine particulate fractions, than in the infection), infected urine, urine filtrate, and E, coli sample containing only the Hepes buffer solution. TLC of particulate fractions particulate fractions from infected urine showed that 82 to 97% When the particulate fraction of urine filtrate was assayed,of the radioactivity migrated with citrulline. The particulate NO significant radioactivity (1900 CPMIIOO s1 effluent) was found in synthase activity was corrected by multiplying '4C-product the column effluent. This activity was inhibited by heating theactivity by the percentage of '4C-citrulline formation measured enzyme but was not inhibited by NO synthase inhibitors. Thus,by TLC (Table 1). In male and in female infected urine pellets, thin layer chromatography was used to determine what percent-the corrected NO synthase activity is 48 9 pmollmin/mg and age of the measured radioactivity migrated with citrulline.99 20 picomollmin/mg, respectively. Corrected NO synthase Smith et a!: NO synthase and nitrite in urinary infection 589

50 80

0C 60 30 0 C C 0 C .0 a) -C 020 40 a) C 0a) 10 a-a) 20 0 Inhibitor,100pM Fig.4. Percent inhibition of particulate NO synthase by NGnitroL arginine (NNA; El), NGmonomethylLarginine (NMA; ), and L-canavanine (•). Results are reported as percent inhibition SEM. 0 10 100 1000

Canavanine,pM activity in noninfected urine from men and women is 0.20.1 Fig.5. Effect of increasing concentrations of L-canavanine on NO and 11 2, respectively. synthase activity. L-canavanine (10, 100, 1000 M) inhibited infected and control urine particulate NO synthase activity 11 2% (N =5),39 Differential inhibition of NO synthase by L-canavanine, 3% (N =8),and 68 4% (N =14),respectively. NGmonomethylLarginine (NMA), and NGnitroLarginine (NNA) NO synthase activity was measured in the presence ofurine. Second, '4C citrulline, isolated by column chromatogra- L-canavanine, NMA, or NNA in infected and control urinephy, is formed from '4C-L-arginine in both soluble and partic- particulate fractions. As shown in Figure 4, L-canavanine,ulate urine fractions. Thin layer chromatography shows that NMA, and NNA (100 LM each) inhibited NO synthase activitybetween 75% and 97% of the radioactivity in female control, 39±3%(N= 16),30±4%(N=5),and5±2%(N8),male infected urine, and female infected urine effluents migrate respectively. The differential inhibition of NO synthase activitywith citrulline. In contrast, only 5 to 13% of the radioactivity in was significant to 95% by the Scheffe F-test. Figure 5 illustratesbacterial effluent migrates with citrulline. Third, 14C-arginine that L-canavanine (10, 100, and 1000 LM) inhibited enzymemetabolism (NO synthase activity) is reduced 94% in the activity in a dose dependent manner [11 1.9% (N =5),39 absence of NADPH and 39% and 30% in the presence of 3.3% (N =8),and 68 4.2% (N =14),respectively]. canavanine (100 M) and NMA (100 MM), respectively. Finally, particulate and soluble NO synthase activities in infected urine NO synthase activity in infected urine depended upon pellet are elevated. NADPH but not calcium Because many studies have confirmed the direct relationship NO synthase activity in particulate fractions did not decreasebetween urine nitrite and urinary tract infection, the Griess significantly when assayed in the presence of 5.0 mM EGTAreagent is used to screen urine for microorganisms. The false without exogenous CaCI2. The average NO synthase activitypositive rate is small (1 to 2%); however, when random urine measurement for male and female infected urine samples in thespecimens are evaluated, the false negative rate can be greater presence of CaCl2 (1.0 mM) was 8114 pmol arginine metab-than 50% [11, 12]. The low sensitivity of the test has been olized!min/mg protein and NO synthase activity in infectedattributed to inadequate bacterial incubation, low urine pH, urine samples without calcium ion was 75 19 pmol arginineurine contents such as urobilinogen, and the finding that some metabolized/mm/mg protein (P =0.84).Similarly, control urinebacteria do not reduce nitrate to nitrite [13]. particulate activity from both women and men did not decrease Roy and Wilkerson [14] found fewer false negative results in the absence of calcium (N =8).Without NADPH, male andwith urine incubation, however, addition of KNO3 to urine female infected urine particulate enzyme activity decreasedsamples in their study did not increase significantly the sensi- 94% from 81 14 to 4.5 0.8 pmol arginine metabolized!tivity of the Griess reaction. In the present experiment, both mm/mg protein (P0.01, N 8). time and the addition of NaNO3 increase nitrite levels in infected urine. Furthermore, L-arginine increases nitrite in Discussion whole infected urine but fails to increase nitrite in urine filtered These results show NO synthase activity in endogenous urineto remove endogenous inflammatory cells. Neither exogenous cells. First, arginine increases nitrite production in wholenitrate nor arginine increase nitrite levels in non-infected urine. infected urine, but not in the bacteria isolated from infectedThese findings suggest that the Griess reagent may be more 590 Smith et a!: NO synthase and nitrite in urinary infection effective in detecting urinary tract infections if urine samplesFinally, the measured difference in NO synthase activity be- are incubated for four hours in the presence of exogenoustween male and female control urine maybe secondary to male arginme. intrinsic NO synthase blocking agents not found in women. The regulation of NO synthase activity in infected urine isTherefore, future studies using tools such as antibody and RNA similar to that of induced forms of activity that have beenprobes are needed to help distinguish the distinct forms of described in rodent macrophages, inflammatory neutrophils,human NO synthase and clarify the etiology of elevated or and carcinoma cells [6]. In our experiment, despite the additiondecreased NO synthase activity in control urine. of NO synthase inhibitors, some '4C-product is still generated. Previous investigators have noted heterogeneity between Also, the TLC experiment reveals citrulline as 82 to 97% ofrodent and human macrophage nitrite production and expressed effluent radioactivity in infected urine, 75% of effluent radioac-concern over defining the role of nitric oxide synthase in human tivity in control female urine, and 28% of effluent radioactivityinflammatory processes based on rodent studies [7]. In these in control male urine. NO synthase is not the only pathway byexperiments, endogenous enzyme activity, from urine particu- which citrulline is generated. In murine macrophages andlate fractions, that converts 14C-argimne to '4C-citrulline is bovine epithelial cells, arginine is metabolized to ornithine byelevated in infected urine, is NADPH dependent, is calcium arginase. Ornithine, in turn, can be converted to citrulline byindependent, and is inhibited more by L-canavanine than by transcarbamoylase [15, 16]. Only a small fraction of arginine isNMA or NNA. These results describe nitric oxide synthase utilized for new protein synthesis while large amounts ofactivity in human inflammatory cells. Understanding the endo- L-arginine are consumed directly to L-ornithine and urea bygenous nitrite source may improve Griess-nitrite screening tests arginase and in activated cells, arginine follows the nitric oxidefor urinary tract infections and lead to distinguishing distinct synthase pathway [15—17]. Arginase is not inhibited by NOforms of human NO synthase. synthase inhibitors and the activity of arginase is similar whether or not macrophages are activated [17]. Acknowledgments The NO synthase activity in infected urine is localized This research is supported by the National Institutes of Health grant primarily (>90%) in the KCI washed particulate fraction fromDK 38311. We would like to thank Dr. Ajay Nangia, Dr. Victor Morris, urine. Previously, inducible NOS has been described as solu-and the emergency room, microbiology laboratory, and urology clinic ble, but recent reports describe NOS activity in RAW264.7staff at Yale-New Haven hospital for their assistance in obtaining urine macrophages that is found in both particulate and solublespecimens. fractions [18]. The enzyme activity in the particulate fraction from infected urine pellet is inhibited to the greatest extent by Reprint requests to Robert M. Weiss, M.D., Section of Urology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New L-canavanine, less by NGmonomethylLarginine (NMA), andHaven, Connecticut 06510, USA. not by N°-nitro-L-arginine (NNA). NO synthase activity in infected urine pellet requires NADPH but is calcium indepen- References dent. Previous studies have shown that NNA preferentially blocks the calcium/calmodulin-dependent NO synthase found 1. CRUICKSHANK J, MOYES JM: The presence and significance of in brain, that NMA exhibits similar potencies against the nitrites in urine. Br Med J 2:712—713, 1914 2, GRIESS P: Bermerkungen zu der Abhandlung der HH. Wesley and calcium/calmodulin-dependent and calciumlcalmodulin-inde- Benedikt Ueber einige Azoverbindungen, Ber Deutsch Chem Gen pendent NO synthases, and that L-canavanine selectively in- 12:426, 1879 hibits the inducible form of enzyme activity that has been found 3. WAGNER DA, YouNG VR, TANNENBAUM SR: Mammalian nitrate in macrophages [19—22]. 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SESSA, WC, HECKER M, MITCHELL JA, VANE JR: The metabolism have been calcium independent and selectively inhibited by of L-arginine and its significance for the biosynthesis of endotheli- L-canavanine [6]. Maybe the NO synthase constitutive/induc- urn-derived relaxing factor: L-Glutamine inhibits the generation of ible dichotomy does not hold in humans. On the other hand, NO L-arginine by cultured endothelial cells. Proc Nat! Acad Sc! USA 87:8607—8611, 1990 synthase activity in female urine may be chronically induced11. POWELL HR, MCCREDIE DA, RITCHIE MA: Urinary nitrite in since about 4% of young women have bacteriuria and the symptomatic and asymptomatic urinary infection. Arch Dis Child percentage of women with bactenuria rises with age [24]. 62:138—140, 1987 Smith et at: NO synthase and nitrite in urinary infection 591

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