Nitrotyrosine in plasma of celiac disease patients as detected by a new sandwich ELISA.

Inflammation is characterized by increased nitric oxide production. Nitrotyrosine has recently been suggested to be useful as a marker for NO-mediated tissue damage. In context of the development of an ELISA for detection of nitrotyrosine in plasma, monoclonal anti-nitrotyrosine antibodies were developed by injecting mice with nitrated keyhole limpet hemocyanin. The specificity of the antibodies was determined by binding to nitrated BSA, lack of binding to unmodified BSA, tyrosine, 3-chlorotyrosine or phenylalanine and inhibition of binding by nitrotyrosine. The antibodies developed are useful for Western blot analysis and immunohistochemical staining. Using these antibodies a nitrotyrosine sandwich ELISA was developed with a lower detection limit of approximately 0.2 nM. The intra- and interassay variance were 2.4% and 11.9%, respectively. Using this newly developed ELISA, 1.27 +/- 1.03 microM nitrotyrosine was detected in plasma samples of celiac disease patients whereas nitrotyrosine was undetectable in control samples. Elevated nitrotyrosine levels were paralleled by an increase in plasma concentrations of NO-oxidation products (NOx), nitrite and nitrate from 15.1 +/- 6.1 microM in controls to 61.0 +/- 28.2 microM in celiac disease patients. Both nitrotyrosine and NOx levels declined when the patients were on a gluten-free diet, suggesting a relation between intestinal inflammation and plasma nitrotyrosine and NOx levels.

Piezogenic papules of the feet in healthy children and their possible relation with connective tissue disorders.

Piezogenic papules (PP) are pressure-induced lesions that appear on the heels while bearing weight, due to herniation of fat tissue into the dermis. They are present in the majority of adults. Because of the poor quality of connective tissue in hereditary disorders of connective tissue, such as the Ehlers-Danlos syndrome, it has been suggested that PP would be larger in number and diameter in this group of disorders. If papules are present, they might be painful as well. In view of this hypothesis 322 healthy pupils aged 4 to 13 years from a Dutch primary school were examined in order to study the prevalence and characteristics of piezogenic papules and signs of connective tissue disorders (Ehlers-Danlos) such as hypermobility and skin fragility. Of the 322 children investigated, 72% had one or more PP, the average number being five, with a mean diameter of 3.3 mm. The mean papule diameter increased with age and body weight. None of the papules were painful. Hypermobile joints occurred in 4.3% of the children. Mean body weight was the same in hypermobile and nonhypermobile children of the same age. The numbers of PP were equal in both groups, as was the number of children with and without PP. None of the children showed skin fragility. Our conclusion is that PP are present in the majority of healthy children, and are never painful.

Increased urinary nitric oxide oxidation products in children with active coeliac disease.

BACKGROUND: Nitric oxide (NO) production catalyzed by iNOS (inducible NO synthase) is thought to take place mainly in macrophages after activation by inflammatory mediators. NO is subsequently oxidized to nitrite and nitrate, which are excreted in urine. The concentration of inflammatory mediators in small bowel biopsy specimens from patients with coeliac disease is increased. The latter could induce increased NO production by stimulation of intestinal macrophage iNOS, resulting in high levels of urinary NO oxidation products, nitrite and nitrate (NOx). AIM: In the present study we evaluated the urinary NOx/creatinine ratios in children with active coeliac disease (n = 22), coeliac disease patients on a gluten-free diet (n = 9), healthy (n = 11) and sick control children (n = 18). METHODS: The Griess reagent method was used for measuring urinary NOx. RESULTS: Median NOx/creatinine ratios of active coeliac disease patients, coeliac disease patients on a gluten-free diet, healthy and sick control patients were 1.21, 0.19, 0.10 and 0.13 mmol/mmol, respectively. All active coeliac disease patients showed increased NOx/ creatinine ratios. Urinary NOx/creatinine ratios of the active coeliac disease patients were significantly higher than those of healthy controls (p < 0.0001), sick controls (p < 0.0001) and coeliac disease patients on a gluten-free diet (p < 0.0001). CONCLUSION: The urinary NOx/creatinine ratio is increased in patients with active coeliac disease and reverts to normal on a gluten-free diet.

Urinary NOx:creatinine ratios during gluten challenge in children with celiac disease.

OBJECTIVES: Celiac disease is a gluten-induced small bowel enteropathy. Inflammation is known to be associated with enhanced nitric oxide (NO) production. An increase in urinary nitrate and nitrite (NOx) reflects increased NO production. The urinary NOx:creatinine ratio can be used as an indicator of the endogenous NO production. The aim of the study was to determine whether the urinary NOx:creatinine ratio of celiac disease patients increases during gluten challenge. METHODS: The authors studied 20 patients with unconfirmed celiac disease who had been following a gluten-free diet for at least 1 year. These patients underwent an 80-day gluten challenge. Urinary samples were obtained before and 10, 20, 40, and 80 days after starting the gluten challenge. The Griess reagent method was used for measuring urinary NOx. RESULTS: Gluten challenge confirmed the diagnosis of celiac disease in 15 of 20 patients. The NOx:creatinine ratios (mmol:mmol) of the biopsy-confirmed celiac disease patients were significantly higher than those of the unconfirmed celiac disease patients (0.67 vs. 0.17 on day 10; 0.78 vs. 0.15 on day 20; 0.85 vs. 0.25 on day 40; and 0.85 vs. 0.17 on day 80). CONCLUSIONS: Gluten challenge resulted in an increased urinary NOx:creatinine ratio in patients with biopsy-confirmed celiac disease. The NOx:creatinine ratio could be useful for the serial evaluation of disease activity.