Serum, Urinary, and Salivary Nitric Oxide in Rheumatoid Arthritis
Nitric oxide (NO) may play important roles in rheumatoid arthritis (RA). RA is an inflammatory disease involving joints and other systems including salivary glands. To assess NO production in RA patients, we compared levels of serum, urine, and salivary nitrite and nitrate (NOx) in patients with RA and normal subjects, and we examined the relationships of these measures to disease activity. Serum, urine, and NOx levels as well as renal creatinine, NOx clearance and fractional excretion rates were compared in 25 RA patients and 20 age- and gender-matched healthy controls. Subjects were hospitalized for 3 days and placed on a NOxrestricted diet. NOx was assayed using nitrate reductase and the Griess reagent. RA activity was assessed using standard clinical and laboratory measures. While consuming a restricted diet for 3 days to eliminate the effects of oral intake of NOx, 24 hour urinary NOx excretion decreased in both RA patients and healthy controls. Urine NOx levels at all time points were not significantly different between RA patients and normal subjects. Serum NOx levels also decreased during the 3 days of NOx restriction, but RA patients had higher serum NOx levels at all time points compared with the control group. Likewise, serum NOx/creatinine ratios were higher in RA patients than in controls. Although basal salivary flow rate and tear flow were lower in RA patients, salivary NOx levels did not differ between normal and RA subjects. While renal creatinine clearance was not different between the two groups, we found that RA patients had lower renal NOx clearance and lower renal NOx fractional excretion. After correction of p values for multiple comparisons, there were no significant relationships for the RA group between measures of disease activity and the urinary NOx, serum NOx, or urinary NOx clearance. Despite interest in the use of NO as a marker of disease activity, alterations in renal NOx clearance and fractional excretion in RA make it difficult to assess in vivo NO production even with strict dietary restriction of NOx intake.
Nitric oxide (NO) is an important mediator of diverse physiologic and pathologic processes, including arthritis. Joint inflammation in autoimmune MRL-lpr/lpr mice and rats with adjuvant-induced arthritis is dependent on the enhanced production of NO. NO, a lipid- and water-soluble gas, is ideally suited as a potent inflammatory mediator because of its strong reactivity with oxygen, superoxide, and iron-containing compounds. This inherent reactivity of NO translates into a relatively short half-life (for example 1 to 10 s), which has made it technically difficult to quantify in solution. Instead of directly measuring NO, investigators have estimated NO production by measuring levels of nitrate (NO3) and nitrite (NO2), stable anions derived from the reaction of NO with superoxide. In general, serum levels and urinary excretion of nitrite + nitrate (NOx) reflect the total production of NO by the body. Care must be taken in the interpretation of results from these studies, because ingested nitrite or nitrate and renal insufficiency elevate both serum and urine nitrate as well as nitrite.
Although previous work has provided evidence in rheumatoid arthritis (RA) for increased production of systemic NO and increased expression of inducible NO synthase (NOS2) and production of NO, most studies of urine and serum NOx levels have been performed in patients eating a normal diet or after only an overnight fast. Other approaches that assess NO production are less subject to dietary influences. For example, nitrotyrosines, which are formed from the reaction of peroxynitrite (a product of NO and superoxide) with tyrosine, can be measured by immunoassay or high-performance liquid chromatography. Using this method, Kaur and Halliwell have detected nitrotyrosines in serum and synovial fluid from patients with active RA, but not in serum from controls.
In the present study, we assessed NO production in vivo by measuring levels of NOx in urine, serum, and saliva in patients with active RA and in normal subjects under conditions of strict dietary NOx restriction. In a comparison between patients with RA and normal subjects, we found that patients with RA had comparable levels of NOx in urine and saliva, elevated serum NOx and serum NOx/creatinine, normal renal creatinine clearance, and reduced renal NOx clearance and fractional excretion. The reduced renal NOx clearance and fractional excretion limit the use of serum NOx and urine NOx excretion as parameters of NO production in patients with RA and their potential as disease markers.
Abstract and Introduction
Abstract
Nitric oxide (NO) may play important roles in rheumatoid arthritis (RA). RA is an inflammatory disease involving joints and other systems including salivary glands. To assess NO production in RA patients, we compared levels of serum, urine, and salivary nitrite and nitrate (NOx) in patients with RA and normal subjects, and we examined the relationships of these measures to disease activity. Serum, urine, and NOx levels as well as renal creatinine, NOx clearance and fractional excretion rates were compared in 25 RA patients and 20 age- and gender-matched healthy controls. Subjects were hospitalized for 3 days and placed on a NOxrestricted diet. NOx was assayed using nitrate reductase and the Griess reagent. RA activity was assessed using standard clinical and laboratory measures. While consuming a restricted diet for 3 days to eliminate the effects of oral intake of NOx, 24 hour urinary NOx excretion decreased in both RA patients and healthy controls. Urine NOx levels at all time points were not significantly different between RA patients and normal subjects. Serum NOx levels also decreased during the 3 days of NOx restriction, but RA patients had higher serum NOx levels at all time points compared with the control group. Likewise, serum NOx/creatinine ratios were higher in RA patients than in controls. Although basal salivary flow rate and tear flow were lower in RA patients, salivary NOx levels did not differ between normal and RA subjects. While renal creatinine clearance was not different between the two groups, we found that RA patients had lower renal NOx clearance and lower renal NOx fractional excretion. After correction of p values for multiple comparisons, there were no significant relationships for the RA group between measures of disease activity and the urinary NOx, serum NOx, or urinary NOx clearance. Despite interest in the use of NO as a marker of disease activity, alterations in renal NOx clearance and fractional excretion in RA make it difficult to assess in vivo NO production even with strict dietary restriction of NOx intake.
Introduction
Nitric oxide (NO) is an important mediator of diverse physiologic and pathologic processes, including arthritis. Joint inflammation in autoimmune MRL-lpr/lpr mice and rats with adjuvant-induced arthritis is dependent on the enhanced production of NO. NO, a lipid- and water-soluble gas, is ideally suited as a potent inflammatory mediator because of its strong reactivity with oxygen, superoxide, and iron-containing compounds. This inherent reactivity of NO translates into a relatively short half-life (for example 1 to 10 s), which has made it technically difficult to quantify in solution. Instead of directly measuring NO, investigators have estimated NO production by measuring levels of nitrate (NO3) and nitrite (NO2), stable anions derived from the reaction of NO with superoxide. In general, serum levels and urinary excretion of nitrite + nitrate (NOx) reflect the total production of NO by the body. Care must be taken in the interpretation of results from these studies, because ingested nitrite or nitrate and renal insufficiency elevate both serum and urine nitrate as well as nitrite.
Although previous work has provided evidence in rheumatoid arthritis (RA) for increased production of systemic NO and increased expression of inducible NO synthase (NOS2) and production of NO, most studies of urine and serum NOx levels have been performed in patients eating a normal diet or after only an overnight fast. Other approaches that assess NO production are less subject to dietary influences. For example, nitrotyrosines, which are formed from the reaction of peroxynitrite (a product of NO and superoxide) with tyrosine, can be measured by immunoassay or high-performance liquid chromatography. Using this method, Kaur and Halliwell have detected nitrotyrosines in serum and synovial fluid from patients with active RA, but not in serum from controls.
In the present study, we assessed NO production in vivo by measuring levels of NOx in urine, serum, and saliva in patients with active RA and in normal subjects under conditions of strict dietary NOx restriction. In a comparison between patients with RA and normal subjects, we found that patients with RA had comparable levels of NOx in urine and saliva, elevated serum NOx and serum NOx/creatinine, normal renal creatinine clearance, and reduced renal NOx clearance and fractional excretion. The reduced renal NOx clearance and fractional excretion limit the use of serum NOx and urine NOx excretion as parameters of NO production in patients with RA and their potential as disease markers.
SHARE
