HARTMUT GRASEMANN

Pulmonary and Critical Care Medicine

FELIX RATJEN

Children's Hospital

	TOTAL SPUTUM NITRATE PLUS NITRITE IS RAISED DURING ACUTE PULMONARY INFECTION IN CYSTIC FIBROSIS

To the Editor:

With great interest we read the recent paper by Linnane and colleagues (1) describing measurements of exhaled nitric oxide (NO) and sputum NO metabolites in cystic fibrosis (CF) patients with acute pulmonary exacerbation. However, there are some issues concerning the interpretation of data that should be addressed.

All CF patients with acute exacerbation had received glucocorticoids before the measurement of exhaled NO. Since glucocorticoids downregulate inducible NO synthase, these measurements are unlikely to reflect spontaneous NO release during acute exacerbation in CF. Alternatively, since exhaled NO has been shown to be positively correlated to lung function in CF patients (2), lower NO concentrations in the CF patients with pulmonary exacerbation may be due to their significantly lower pulmonary function.

NO₂/NO₃ concentrations in saliva are significantly higher in CF patients with exacerbation than in stable CF (3). Expectorated sputum is inevitably contaminated with saliva; high sputum NO metabolite concentrations may therefore reflect increased NO formation in the upper airways rather than in the lung. The validity of comparing spontaneously expectorated sputum with hypertonic saline-induced sputum may be limited since different dilutions of airway secretions may influence final concentrations. Furthermore, sputum nitrate concentrations are positively correlated to sputum rigidity, and differences in sputum NO₂/NO₃ concentrations may, therefore, be the result of changes in viscoelastic properties of CF airway secretions during exacerbation (4).

Nitrate and nitrite concentrations in airway secretions are both influenced by pathogens that may utilize nitrogen oxides for anaerobic metabolism. The airways of the CF patients treated for pulmonary exacerbation in the paper of Linnane and colleagues were all colonized with Pseudomonas aeruginosa, which is able to metabolize both nitrate and nitrite by enzymatic reduction. Concentrations of these two NO metabolites may be affected differently. In a recent study we demonstrated that during treatment for CF exacerbation, sputum nitrite remained unchanged, while nitrate concentrations increased significantly (3). This increase in sputum nitrate concentrations could possibly be explained by a reduction in the bacterial load, resulting in decreased bacterial metabolism of nitrate.

In conclusion, we agree with Linnane and coworkers that exhaled NO is not a useful marker of airway inflammation in glucocorticoid-treated CF patients undergoing acute pulmonary exacerbation. However, due to the complexity of nitrogen oxide metabolism in the airways of patients with CF, sputum NO₂/NO₃ concentrations may not reflect NO synthase activity in the lower airways.

Brigham and Women's Hospital

Harvard Medical School

Boston, Massachusetts

University of Essen

Essen, Germany

	References

1. Linnane, S. J., V. M. Keatings, C. M. Costello, J. B. Moynihan, C. M. O'Connor, M. X. FitzGerald, and P. McLoughlin. 1988. Total sputum nitrate plus nitrite is raised during acute pulmonary infection in cystic fibrosis. Am. J. Respir. Crit. Care Med. 158: 207-212 [Abstract/Free Full Text].

2. Grasemann, H., E. Michler, M. Wallot, and F. Ratjen. 1997. Decreased concentration of exhaled nitric oxide (NO) in patients with cystic fibrosis. Pediatr. Pulmonol. 24: 173-177 [Medline].

3. Grasemann, H., I. Ioannidis, R. P. Tomkiewicz, H. de Groot, B. K. Rubin, and F. Ratjen. 1998. Nitric oxide metabolites in cystic fibrosis lung disease. Arch. Dis. Child. 78: 49-53 [Abstract/Free Full Text].

4. Grasemann, H., R. P. Tomkiewicz, I. Ioannidis, O. E. Ramirez, B. K. Rubin, and F. Ratjen. 1997. Metabolites of nitric oxide and viscoelastic properties of airway secretions in cystic fibrosis (abstract). Am. J. Respir. Crit. Care Med. 155: A46 .

From the Authors:

In their letter, Drs. Grasemann and Ratjen raise several points regarding the concentrations of nitrogen oxides in exhaled gases and in sputum from patients with cystic fibrosis (CF).

They suggest that since the patients were receiving glucocorticoid therapy at the time at which measurements were made, changes in NO and NO metabolites did not result from alterations in inducible nitric oxide synthase (iNOS) activity. This interpretation is based on the assumption that high-dose glucocorticoid therapy invariably suppresses iNOS expression in humans. This is not the case and, in particular, it has been demonstrated that glucocorticoids do not suppress iNOS activity in the airway epithelium (1). Furthermore, in the absence of steroid therapy, NO₃/NO₂ concentrations may have been even higher in our patients, i.e., our data are compatible with partial suppression of iNOS expression. A second possibility is that endothelial nitric oxide synthase (eNOS) activity, which was insensitive to glucocorticoid therapy, was upregulated in the presence of inflammation (2, 3; see DISCUSSION in our article [4]). Thus, we do not agree that the failure of glucocorticoids to lower sputum NO₃/NO₂ concentrations excludes increased NOS activity as the source of these nitrogen oxides during infective exacerbations.

Drs. Grasemann and Ratjen write that "lower NO concentrations in the CF patients with pulmonary exacerbations may be due to their significantly lower pulmonary function." In contrast, we did not find any significant reduction in exhaled NO in our acutely infected group compared to stable CF patients (see Figure 1 in our article [4]).

Drs. Grasemann and Ratjen also suggest that increased salivary concentrations of NO metabolites may have caused the increased concentration of NO₃/NO₂ that we had reported in the sputum of patients with acute exacerbations. This suggestion contradicts their previously published data demonstrating that total salivary NO metabolites are not elevated in CF patients undergoing acute infective exacerbations when compared to stable patients (see Figure 2 in Reference 5). Thus, the influence of any salivary contamination would be to reduce NO₃/NO₂ concentrations in acutely infected patients, not elevate them.

Drs. Grasemann and Ratjen question the validity of comparing spontaneously expectorated and induced sputum. It has been demonstrated that induction of sputum with hypertonic saline does not alter cell counts or fluid phase indices when compared with spontaneously expectorated sputum (6). Furthermore, the concentrations of NO₃/NO₂ that we detected in the induced sputum from control subjects are similar to those that we and others have previously reported in airway epithelial lining fluid collected by bronchoscopy (7, 8). Thus the NO₂/NO₃ concentrations that we report provide a reliable indication of the concentrations in lower airway fluids.

Grasemann and coworkers (9) have previously presented preliminary data suggesting that sputum NO metabolites are positively correlated with sputum viscosity. We have not had an opportunity to examine these data in detail but are unaware of any evidence that increased sputum viscosity causes significant increases of NO₃/NO₂ in patients with CF.

Although Pseudomonas aeruginosa organisms can reduce both nitrate and nitrite under certain conditions, Graseman and colleagues have shown that sputum concentrations of NO metabolites in Pseudomonas-positive and -negative patients are similar (5). This finding suggests that denitrification by this organism does not significantly alter NO₃/NO₂ concentrations in the airway fluid in vivo.

We agree that the concentration of a metabolite in body fluids or tissues at a single point in time is only partially dependent on the rate of production of that metabolite. Nonetheless, sputum concentrations of NO metabolites and the concentrations of exhaled NO have been shown to increase with increasing NOS activity in a wide variety of lung diseases, therefore providing indirect indices of NOS activity in the airway (7, 10, 11). We are unaware of data directly demonstrating an alternative source of nitrogen oxides in the lungs of CF patients.

Paul McLoughlin

Vera Keatings

Muiris X. FitzGerald

Department of Physiology

Department of Medicine and Therapeutics

University College Dublin, Ireland

	References

1. Lundberg, J. O. N., N. E. Weitzberg, J. Rinder, et al . 1996. Calcium- independent and steroid-resistant nitric oxide synthase activity. Eur. Respir. J. 9: 1344-1347 [Abstract].

2. Belvisi, M., P. J. Barnes, S. Larkin, et al . 1995. Nitric oxide synthase activity is elevated in inflammatory lung disease in humans. Eur. J. Pharmacol. 283: 255-258 [Medline].

3. Saleh, D., P. J. Barnes, and A. Giaid. 1997. Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 155: 1763-1769 [Abstract].

4. Linnane, S. J., V. M. Keatings, C. M. Costello, et al . 1998. Total sputum nitrate plus nitrite is raised during acute pulmonary infection in cystic fibrosis. Am. J. Respir. Crit. Care Med. 158: 207-212 .

5. Grasemann, H., I. Ioannidis, R. P. Tomkiewicz, H. de Groot, B. K. Rubin, and F. Ratjen. 1998. Nitric oxide metabolites in cystic fibrosis lung disease. Arch. Dis. Child. 78: 49-53 .

6. Pizzichini, M. M. M., T. A. Popov, A. Efthimiadis, et al . 1996. Spontaneous and induced sputum to measure indices of airway inflammation in asthma. Am. J. Respir. Crit. Care Med. 154: 866-869 [Abstract].

7. Gaston, B., J. Reilly, J. M. Drazen, et al . 1993. Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proc. Natl. Acad. Sci. U.S.A. 90: 10957-10961 [Abstract/Free Full Text].

8. O'Donnell, D., V. Keatings, J. Moynihan, et al . 1997. Exhaled nitric oxide and BAL nitrite/nitrate in active pulmonary sarcoidosis. Am. J. Respir. Crit. Care Med. 156: 1892-1896 [Abstract/Free Full Text].

9. Grasemann, H., R. P. Tomkiewicz, I. Ioannidis, O. F. Ramirez, B. K. Rubin, and F. Ratjen. 1997. Metabolites of nitric oxide and viscoelastic properties of airway secretions in cystic fibrosis. Am. J. Respir. Crit. Care Med. 155: A46 .

10. Barnes, P. J., and M. C. Belvisi. 1993. Nitric oxide and lung disease. Thorax 48: 1034-1043 [Free Full Text].

11. Gaston, B., J. M. Drazen, J. Loscalzo, and J. S. Stamler. 1994. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149: 538-551 [Abstract].