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Keywords:

  • ASA;
  • chronic urticaria;
  • double-blind placebo-controlled;
  • food additive;
  • urinary LTE4;
  • urinary methylhistamine

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

Background: The recovery of mediator metabolites from urine has the potential to provide a rapid, safe, and easily available index of release of mediators. We aimed to determine urinary metabolites of both histamine and leukotrienes (LTs) in patients affected by chronic urticaria (CU).

Methods: Twenty patients with CU were studied. They were selected on the basis of double-blind placebo-controlled challenge (DBPC) with acetyl salicylic acid (ASA) and food additives. Ten patients (group B) were negative to both challenges. Ten patients (group C) presented urticaria and/or the appearance of angioedema during or 24 h after challenge, with reactions to ASA (five patients) or food additives (five patients). We recruited 15 healthy volunteers as controls (group A). During a second challenge, groups B and C were challenged double-blind with a single dose of ASA, or a specific food additive, or placebo. The healthy group was challenged only with a placebo (talc capsule). Patients in groups B and C were challenged twice: with placebo (as groups B1 and C1) and with ASA (groups B2 and C2) or food additives (C2). Four samples of urine were collected; one during the night before the specific or sham challenge (baseline), and three at 2, 6 and 24 h after the challenge. Urinary methylhistamine (N-MH) and LTE4 were analyzed and normalized for urinary creatinine.

Results: For urinary N-MH at baseline, there was a significant difference only between group A and groups B1, B2, C1 and C2 (A vs. B1, P < 0.0001; A vs. B2, P < 0.0001; A vs. C1, P < 0.0001; A vs. C2, P < 0.0001). We detected a significant variation in urinary methylhistamine excretion only in group C2 after 2 h, 6 h and 24 h (P < 0.0001). However, no variations were observed in N-MH excretion rate in the other groups (A, B1, C1) after challenge with placebo, and in B2 after challenge with ASA 20 mg. For urinary LTE4 at baseline no differences were found between the mean values for the different groups. After specific challenge, only C2 patients showed significantly increased excretion rates of urinary LTE4 compared with the other groups challenged with placebo (A, B1, C1), or ASA (B2) (P < 0.0001). No significant correlation was seen between urinary LTE4 and methylhistamine excretion rate in any patients.

Conclusion:  Our results show that urinary excretion of N-MH and LTE4 is different for CU patients without ASA or food hypersensitivity, compared to those with CU with ASA or food additive hypersensitivity after specific challenge.

The recovery of mediator metabolites from urine has the potential to provide a rapid, safe, and easily available index of synthesis or release of mediators as histamine and leukotrienes (LTs). The excretion rates of some metabolites such as those of histamine are known to vary over the course of a day, whereas the excretion of others such as those of leukotriene are relatively constant (1–5).

Blood histamine is rapidly degraded and N-methylhistamine (N-MH) in urine accounts for approximately 10% of histamine catabolism; it is generally considered to be a unique metabolite of histamine (6). In patients with anaphylactic reactions, determination of urine N-MH has been suggested as a useful parameter for showing in vivo histamine release (7).

LTE4 is a stable product of metabolism of cysteinyl LTs. It is excreted in urine in relatively constant proportions. Therefore, the urinary concentration has been used as an integrated measure of overall LT production in the body, in studies concerning both spontaneous and induced attacks of asthma (8–12). In fact, urinary LTE4 excretion rates are known to increase during the early-phase response after allergen challenge, and aspirin exposure causes a striking increase in urinary LTE4 excretion in patients with aspirin-sensitive asthma (11).

Chronic urticaria (CU) is conventionally defined as the daily—or almost daily—occurrence of urticarial wheals for at least 6 weeks with or without angioedema. Its etiology still remains unclear despite great efforts, and patients must be managed symptomatically. The key cell in CU is the dermal mast cell, and any hypothetical etiologic mechanism should explain how this cell becomes repeatedly and extensively activated, leading to release of histamine and the other mediators. However, other cell types are involved also, including the basophil (13–16).

Most patients feel at some time that food “allergy” is causative. Certainly IgE-mediated reactions caused by foods are important causes of acute allergic urticaria, but they can rarely, if ever, be substantiated as a cause of CU. On the other hand, idiosyncratic reactions to food additives have been suggested by a number of authors to be important causes (17,18). About 30% of patients with CU experience flares of hives and/or angioedema after ingesting acid acetylsalicylic (ASA) (19). The mechanisms of such reactions are still poorly defined. ASA hypersensitivity suggests a mechanism that depends on cyclooxygenase inhibition, with increased 5′-lipoxygenase-mediated production of leukotrienes (19–21). It is likely that the hypersensitivity to food additives, which characterizes a subset of people with CU, has pathogenic mechanisms similar to those hypothesized for ASA hypersensitivity. The diagnosis of food hypersensitivity to ASA and/or food additives is a positive placebo-controlled challenge testing. The hypothesis that LTs are important in CU is supported by some recent reports of the effectiveness of LT antagonists in some people with chronic urticaria (22–26). However, the role of LTs in CU is not yet clear, although they are known to have potent local effects on cutaneous vasculature, and are able to cause a wheal and erythema response in human skin (27).

In this study, we collected four consecutive urine specimens from 15 normal people and 20 with stable mild-to-moderate chronic urticaria, before and after challenge, to measure the excretion rates of urinary N-MH and LTE4. Our data show no significantly variable rates of either urinary N-MH or LTE4 excretion after sham (placebo) challenge, either in healthy people or in patients with CU. A significant increase of both mediators was seen only after a specific challenge with ASA or a food additive. Urinary assessment of LTE4 might, therefore, be a useful marker when selecting drugs for patients affected by chronic urticaria who have hypersensitivity to ASA and food additives.

Subjects and diagnostic protocols

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

Twenty people with chronic urticaria were selected by chart review from outpatients of the Department of Clinical and Experimental Medicine of Verona (Italy). All of those selected had been previously submitted to oral challenges for the following chemical substances (graded doses in brackets): acetyl salicylic acid/aspirin (10, 10, 20 mg), tartrazine (10, 10, 20 mg), erythrosine (50, 50, 100 mg), monosodium benzoate (50, 50, 100 mg), p-hydroxybenzoate (50, 50, 100 mg), sodium metabisulfite (5, 5, 10 mg), monosodium glutamate (100, 100, 200 mg). All challenges were double-blind placebo-controlled (DBPC).

On the basis of the DBPC, 10 patients appeared hypersensitive, five to ASA (10 mg), two to tartrazine 20 mg, and one each to sodium benzoate (100 mg), sodium metabisulfite (10 mg), and monosodium glutamate (200 mg) (26). No other patient affected by chronic urticaria presented unequivocal urticaria (defined as pruritic and erythematous areas raised over normal skin) and/or the appearance of angioedema (defined as swelling of skin and/or external mucosa) during DBPC or after 24 h. According to test results, patients affected by chronic urticaria without any reactions to ASA or food additives were defined as those without positivity to challenge (group B). Those affected by chronic urticaria with reactions to ASA or food additives were defined as those with positivity to ASA or food additives (group C). As a control group (group A) we recruited 15 healthy volunteers from the staff of the department; they had no history of urticaria, rhinitis, asthma, atopic eczema/dermatitis syndrome (AEDS) (32), or other relevant diseases.

Study protocol

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

The Ethics Committee of Department of Clinic and Experimental Medicine of Verona (Italy) approved the study. All participants gave written consent. Based on the results of previous challenges to food additives and ASA (see above) the patients with CU and hypersensitivity to ASA or food additives (group C) were challenged double-blind with a single dose of ASA or specific food additive, inducing urticaria, or placebo. Patients without previous challenge positivity to food additives or ASA (group B), and healthy subjects (group A), were challenged with only placebo (talc capsule). The patients in groups B and C were challenged twice, on different days, forming groups B1 and C1 when they were challenged with placebo, and B2 and C2 when challenged with ASA (B2 and C2) or food additives (C2). None of the patients showed significant cutaneous symptoms at the time of testing. Antihistamines were interrupted at least 8 days before the challenges, and no drugs were used within 48 h of testing. No patients had taken oral corticosteroids.

Four urine samples were collected for each challenge, one during the night before the specific or sham challenge (baseline), and three at 2 h, 6 h, and 24 h after challenge (groups B2, C2) or sham-challenge (groups A, B1, C1). Urine samples were collected in polystyrene vessels, volumes were determined, and aliquots were immediately frozen at −80°C.

N-MH determination in urine

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

N-MH in urine was measured by a double-antibody radioimmunoassay (RIA) according to manufacturer's instructions (Pharmacia, Uppsala, Sweden). Urine samples were thawed and diluted 1 : 51 with phosphate buffered saline; 200 µl of diluted urine or N-MH standards were mixed with the same amount of 125I-labeled histamine, coupled to dog serum albumin. Then 200 µl of N-MH-specific monoclonal antibody (mAb) was added, and the reaction mixture was incubated overnight (16–20 h) in refrigerator (+2 to +8°C). Thus, N-MH in the sample was competing with a fixed amount of 125I-labeled histamine albumin complex for the binding sites of the specific mAb (ratio of N-MH : histamine specificity ∼ 18 : 1). The next day, bound and free N-MH were separated by adding a second immunoadsorbent antibody Sepharose antimouse IgG (raised in sheep). Incubation was performed for 30 min at room temperature. After centrifugation, tubes were decanted, and radioactivity in the pellet was measured. Resulting counts per minute from 0.2 to 10 ng/mg of N-MH standard solutions were to construct standard curves. Concentrations of unknown samples were determined by comparison. According to instructions supplied by the manufacturer, assay characteristics were: detection limit < 0.1 ng/mg; measuring range 0.2–10 ng/mg; recovery on addition 86% to 117% assay specificity at 50% uptake for N-MH 100%, histamine 5.6%, serotonin and histidine < 0.005%(data based on study performed at Pharmacia Diagnostics AB). All measurements were made in duplicate. N-MH excretion rates were normalized using urinary creatinine concentrations and expressed as ng/mmol creatinine; creatinine concentrations were determined according to Titz (28).

LTE4 determination in urine

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

LTE4 determination in urine was performed exactly as described by Bellia et al. (12). Urine samples were thawed and to each 5 mg aliquot, 4000 c.p.m. of tritiated-LTE4 was added (Du Pont de Nemours Italiana-NEN Products, Cologno M, Italy). The sample was loaded into an LC18 cartridge (Supelco, Bellefonte, PA), preconditioned with methanol 100% (10 mg), distilled water (10 mg), and formic acid at pH 3. Cysteinyl LTs were eluted with methanol 80% and water at pH 6; this fraction was then evaporated to dryness and reconstituted with 500 µl of aqueous methanol (40%). Separation of LTE4 was performed by high-performance liquid chromatography with System Gold apparatus (Beckman Instruments, Fullerton, CA) equipped with a UV168 detector, an RP-18 precolumn (Waters Associates, Milford, MA), and an Ultrasphere 25 cm octadecylsilyl 5 µm column (Beckman). The gradient system included two solvents: solvent A, 50 : 50 : 0.02 methanol : water : acetic acid at pH 5.5 with ammonium hydroxide; and solvent B, methanol 100%. A flow rate of 1 mg min−1 was used. Under these conditions LTE4 was eluted at 25–30 min. Thirty-five 1 mg fractions were collected and dried, and 200 µl of each was used to assess recovery and LTE4 elution by measuring radioactivity (LS1801 γ-counter, Beckman). Eight hundred ml of fractions containing tritiated LTE4 and the three fractions eluted before and after the peak were evaporated and redissolved in 250 µl of radioimmunoassay buffer. Then LTE4 was measured by a radioimmunoassay commercial kit (Amersham Italia, Milan, Italy). LTE4 was the radioligand, and samples were quantified against an LTE4 standard curve with a mAb for LTC4 that had good cross-reactivity with LTE4. All measurements were made in duplicate, corrected for recovery (45% to 65%), and expressed in ng/mmol of creatinine. Previously established inter- and intra-assay coefficients of variability were 12% and 7%, respectively; sensitivity was 8 pg. LTE4 excretion rates were normalized with the use of urinary creatinine concentrations (28).

Statistical analysis

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

The results were expressed as mean ± SD for convenience. Because the distribution of data was not normally distributed, nonparametric tests were used for statistical analysis. A Kruskal–Wallis nonparametric test was used to compare between groups. The Mann-Whitney test was used to analyze the significance of differences in values of urinary methylhistamine and LTE4 obtained from patients of group C after challenge with placebo and a specific additive. Finally, we used this test to analyze the significance of differences of urinary N-MH and LTE4 in patients intolerant to ASA and those intolerant to food additives. Spearman's rank correlation test (rho, and 95% confidence interval (CI) for rho) was performed to assess whether urinary methylhistamine and LTE4 were related. All analyses were performed using StatDirect (Tidestone Technologies, UK) on a PC. P values of ≤ 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

The characteristics of the participants are reported in Table 1. The three groups did not differ with respect to age and sex. Groups B and C did not differ with respect to the onset of urticaria. No subject had adverse reactions to sham challenge. After specific challenge, all of group C (but not of group B) had reactivation of urticaria after 60–90 min. On the basis of results of the diagnostic protocol, ASA-sensitive patients (n = 5) were challenged with 10 mg ASA, tartrazine-sensitive (n = 2) with 20 mg, sodium benzoate-sensitive (n = 1) with 100 mg, sodium metabisulfite-sensitive (n = 1) with 10 mg, and monosodium glutamate-sensitive (n = 1) with 200 mg. The patients of group B were challenged with placebo (B1) and with 20 mg ASA (B2).

Table 1.  Characteristics of participants
 Healthy subjectsChronic urticaria patients
Without intoleranceWith intolerance*
  • *

    Intolerant to acetyl salicylic acid (ASA) or food additives. Patients sensitive to ASA ( N  = 5), tartrazine ( N  = 2), sodium benzoate ( N  = 1), sodium metabisulfite ( N  = 1), monosodium glutamate ( N  = 1).

  • NA = Not applicable.

GroupGroup AGroup BGroup C
Age (years ± SD)41.4 ± 14.841.1 ± 14.340.7 ± 14.3
Sex (male/female)6/94/64/6
Onset of urticaria (months ± SD)NA98.0 ± 67.188.1 ± 65.2

Urinary N-HM

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

Significant differences were found between the groups for urinary N-MH (calculated as ng/mmol urinary creatinine).

At baseline, the mean values of N-MH were 3.4 ± 1.8 ng/mmol for group A, 15.8 ± 3.3 ng/mmol for B1, 15.6 ± 3.3 for B2, 15.9 ± 3.3 ng/mmol for C1, and 16.1 ± 3.5 ng/mmol for C2 (A vs. B1, P < 0.0001; A vs. B2, P < 0.0001; A vs. C1, P < 0.0001; A vs. C2, P < 0.0001; B1 vs. C1, P = NS; B1 vs. C2, P = NS; B2 vs. C1, P = NS; B2 vs. C2, P = NS; B1 vs. B2 P = NS; C1 vs. C2, P = NS).

After 2 h, the mean values were 3.7 ± 2.1 ng/mmol for group A, 15.6 ± 3.7 ng/mmol for B1, 15.5 ± 3.4 ng/mmol for B2, 15.9 ± 3.3 ng/mmol for C1, and 65.2 ± 11.3 ng/mmol for C2 (B1 vs. A, P < 0.0001; B2 vs. A, P < 0.0001; C1 vs. A, P < 0.0001; C2 vs. A, P < 0.0001; B1 vs. C1, P = NS; B2 vs. C1, P = NS; C2 vs. B1, P < 0.0001; C2 vs. B2, P < 0.0001; B1 vs. B2 P = NS; C2 vs. C1 P < 0.0001).

After 6 h, the mean values were 3.4 ± 1.4 ng/mmol for group A, 15.7 ± 3.7 ng/mmol for B1, 11.0 ± 3.3 ng/mmol for B2, 16.0 ± 0.9 for C1, and 65.2 ± 3.8 ng/mmol for C2 ( B1 vs. A, P < 0.0001; B2 vs. A, P < 0.0001; C1 vs. A, P < 0.0001; C2 vs. A, P < 0.0001; B1 vs. C1, P = NS; B2 vs. C1, P = NS; C2 vs. B1, P < 0.0001; C2 vs. B2, P < 0.0001; B1 vs. B2, P = NS; C1 vs. C2, P = 0.0001).

After 24 h, the mean values were 3.5 ± 1.5 ng/mmol for A, 14.9 ± 2.8 ng/mmol for B1, 15.8 ± 3.2 ng/mmol for B2, 15.7 ± 2.7 ng/mmol for C1, and 59.9 ± 14.4 ng/mmol for C2 (B1 vs. A, P < 0.0001; B2 vs. A, P < 0.0001; C1 vs. A, P < 0.0001 C2 vs. A, P < 0.0001; C1 vs. B1, P = NS; C1 vs. B2, P = NS; C2 vs. B1, P < 0.0001; C2 vs. B2, P < 0.0001; B1 vs. B2, P = NS; C2 vs. C1 P < 0.0001).

Individual values of urinary N-MH for groups C1 and C2 are given in Fig. 1A and 1B. Regarding intragroup differences, significant differences were observed between the values of N-MH at baseline vs. 2 h, 6 h, and 24 h (P < 0.0001) only in group C2, after challenge with ASA or food additives (Fig. 2). In group C2 we found no significant differences between patients hypersensitive to ASA and those to food additives (Table 2).

image

Figure 1. Individual excretion ratio of urinary methylhistamine (N-HM) before (0) and 2 h, 6 h and 24 h after challenge, normalized for urinary creatinine. (A) The individual excretion ratio of urinary N-HM in patients with chronic urticaria (CU) after challenge with placebo (group C1; P  = NS between the values at any time of the study). (B) The individual excretion ratio of urinary N-HM in patients with CU after challenge with acetyl salicylic acid (ASA) or specific food additive, inducing urticaria (group C2; P  = 0.0001 between the values of N-MH at baseline vs. 2 h, 6 h and 24 h).

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image

Figure 2. Means (± SD) of excretion ratio of urinary methylhistamine (N-HM) in patients with chronic urticaria (CU) before and after challenge with placebo (group C1; see text) and before and after challenge with acetyl salicylic acid (ASA) or a specific food additive, inducing urticaria (group C2; see text) normalized for urinary creatinine. There were no significant differences in group C1 at all time-points of the study, whereas group C2 showed significant differences between the means of prechallenge excretion ratio and any time-points of the study. No significant differences were seen between the means of N-HM after specific challenges in group C2.

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Table 2.  Methylhistamine (M-HN) and leukotriene E 4 (LTE 4 ) in the urine of patients affected by chronic urticaria, hypersensitive to acetyl salicylic acid (ASA) or food additives (group C)
 M-HN (ng/mmol of creatinine)LTE4 (ng/mmol of creatinine)
ASA*AdditivesPASA*AdditivesP
  • *

    Five patients had a challenge positive to ASA (see Table 1).

  • Five patients had a challenge positive to food additives (see Table 1).

  • P values refer to the difference between the M-HN or LTE 4 urinary values of patients hypersensitive to ASA or additives.

Baseline15.8 ± 1.416.4 ± 4.20.516.8 ± 0.917.4 ± 1.300.8
After 2 h65.6 ± 7.564.7 ± 6.70.935.9 ± 1.150.9 ± 6.5 0.09
After 6 h64.8 ± 3.865.5 ± 7.00.840.7 ± 2.940.9 ± 5.800.6
After 24 h56.3 ± 5.863.6 ± 1.30.426.4 ± 2.643.3 ± 7.800.1

Urinary LTE4

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

At baseline, no differences were found between the mean values of urinary LTE4 (calculated as ng/mmol urinary creatinine), the different s. In group A mean values were 16.8 ± 2.3 ng/mmol, 16.2 ± 2.1 ng/mmol in B1, 16.1 ± 2.2 ng/mmol in B2, 17.2 ± 2.4 ng/mmol in C1, and 17.1 ± 2.6 ng/mmol in C2.

At all the times no differences were found between mean values for group s A, B1, B2 and C1 (after 2 h, A 16.6 ± 2.4 ng/mmol, B1 16.4 ± 3.2 ng/mmol, B2 16.4 ± 2.5 ng/mmol, and C1 17.1 ± 2.6 ng/mmol; after 6 h, A 16.9 ± 3.1 ng/mmol, B1 16.1 ± 2.2 ng/mmol, B2 16.4 ± 3.2 ng/mmol, and C1 17.2 ± 2.4 ng/mmol; after 24 h, A 16.7 ± 2.5 ng/mmol, B1 16.1 ± 2.3 ng/mmol, B2 16.4 ± 2.3 ng/mmol, and C1 16.9 ± 2.2).

In these groups, there were no significant differences between the values of urinary LTE4 at baseline vs. 2 h, 6 h and 24 h after the sham challenge and after challenge with ASA in B2. In Fig. 3A and 3B the individual values of urinary LTE4 in groups C1 and C2 are reported. The mean values of C2 after specific challenge with ASA or with food additive were significantly higher (43.4 ± 12.7 after 2 h, 40.8 ± 9.8 after 6 h, and 34.9 ± 15.2 after 24 h) than those of the other groups (after 2 h: C2 vs. A, P < 0.0001, C2 vs. B1, P < 0.0001, C2 vs. B2, P < 0.0001, C2 vs. C1, P < 0.0001; after 6 h: C2 vs. A, P < 0.0001, C2 vs. B1, P < 0.0001, C2 vs. B2, P < 0.0001, C2 vs. C1, P < 0.0001; after 24 h, C2 vs. A, P = 0.0004, C2 vs. B1, P < 0.0001, C2 vs. B2, P < 0.0001, C2 vs. C1, P < 0.0001). In group C2 significant differences (P < 0.0001) were observed between the values of urinary LTE4 at baseline vs. 2 h, 6 h and 24 h after challenge with ASA or food additives (Fig. 4). Group C2 showed no significant differences between patients with ASA hypersensitivity and those with intolerance to food additives (Table 2). We found no significant correlations between urinary N-MH and LTE4 values at all time-points in all the groups.

image

Figure 3. Individual excretion ratio of urinary leukotriene LTE 4 before (0) and 2 h, 6 h and 24 h after challenge, normalized for urinary creatinine. (A) Individual excretion ratio of urinary LTE 4 in patients with chronic urticaria (CU) after challenge with placebo (group C1; P  = NS between the values at any time of the study). (B) Individual excretion ratio of urinary LTE 4 in patients with CU after challenge with acetyl salicylic acid (ASA) or a specific food additive, inducing urticaria (group C2; P  = 0.0001 between the values of LTE 4 at baseline vs. 2 h, 6 h and 24 h).

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image

Figure 4. Means (± SD) of excretion ratio of urinary leukotriene(LT)E 4 in patients with chronic urticaria (CU) before and after challenge with placebo (group C1; see text) and before and after challenge with acetyl salicylic acid (ASA) or a specific food additive, inducing urticaria (group C2; see text) normalized for urinary creatinine. There were no significant differences in group C1 between all time-points of the study, whereas group C2 showed significant differences between the means of prechallenge excretion ratio and any time-points of the study. No significant differences were seen between the means of LTE 4 after the specific challenges in group C2.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References

Hypersensitivity to ASA and food additives may be a subtle cause of chronic urticaria (14, 29–32). There are no in vitro tests to identify patients with ASA- or food additive-sensitive urticaria. Thus, oral challenges of ASA or food additives are the only way to identify ASA- or food additive-induced cutaneous reactions (18, 32, 33).

The prevalence of reactions to food additives in people with chronic urticaria is unknown, but the prevalence of ASA-induced urticaria in those with chronic urticaria has been reported to vary between 21% and 30%. In fact, the difference in prevalence might depend on the kind of clinic performing the study (33,34). However, approximately 15% of aspirin-sensitive individuals are sensitive to tartrazine yellow no. 5 and an investigation of well-documented tartrazine-sensitivity in patients with intractable chronic urticaria reported an incidence rate of 8% (35, 36).

The pathogenesis of urticaria should involve the release of potential vasoactive mediators that arise from the mast cells (14). A wide variety of substances (drugs, contrast media, and food additives) have been shown to activate mast cells or basophils, or both, and mediator release should account for the hypersensitivity-inducing effects of these agents. In general, such substances appear to have direct histamine-releasing properties that are unlikely to be mediated via specific IgE molecules (37), and the release of histamine, leukotrienes and other mediators induced by challenge with some additives (e.g. ASA and sulfites) were not IgE-mediated (38,39) On the other hand, the LTs release from various cell types may be induced by agonists (40). Accordingly, the mechanisms of hypersensitivity to additives remain largely unknown.

The results of this study indicate that after specific challenge urticaria exacerbations are accompanied by an increased release of N-MH and cysteinyl LTs. On the other hand, chronic urticaria seemingly not linked to responses to food additives or ASA is characterized by increased baseline production of histamine, but not of leukotrienes. The spontaneous increase of NM-H in urine of chronic urticaria patients has been previously reported by other groups (14), so the most important data from this study is that only the patients with ASA- or food additive-hypersensitivity, after the specific challenge, present a higher urine concentration of both histamine and LTs. Accordingly, it has been suggested that LTs are important in pathogenesis of chronic urticaria, by triggering its clinical symptoms (erythema, angioedema, itching); this suggestion is strengthened by the observation that leukotriene receptor antagonists may be effective in chronic urticaria (22–26). However, the complexity of this matter is demonstrated by a report suggesting that the pathogenesis aspirin-induced urticaria may depend on stimulation of LT receptors (41).

In conclusion, our results point to the role of both histamine and LTs in chronic urticaria with people with hypersensitivity to food additives and ASA. However, the fact that these substances are released in increased amounts is not definitive evidence of a biological role. Indeed, attenuation or elimination of the positive response to challenge with relevant antagonists would have to be shown. Nonetheless, the present results suggest that the determination of urinary LTE4 may be used as an additional diagnostic tool.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects and diagnostic protocols
  5. Study protocol
  6. N-MH determination in urine
  7. LTE4 determination in urine
  8. Statistical analysis
  9. Results
  10. Urinary N-HM
  11. Urinary LTE4
  12. Discussion
  13. Acknowledgments
  14. References