Discovery of a Potential Human Serum Biomarker for Chronic Seafood Toxin Exposure Using an SPR Biosensor.

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 µg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers' urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern.

Perioperative Exposure to Suspect Neurotoxicants From Medical Devices in Newborns With Congenital Heart Defects.

BACKGROUND: Industrial chemicals are increasingly recognized as potential developmental neurotoxicants. Di(2-ethylhexyl) phthalate (DEHP), used to impart flexibility and temperature tolerance to polyvinylchloride, and bisphenol A (BPA), used to manufacture polycarbonate, are commonly present in medical devices. The magnitude of exposure in neonates during hospitalization for cardiac operations is unknown. METHODS: We quantified urinary concentrations of DEHP metabolites and BPA preoperatively and postoperatively in neonates undergoing cardiac operations and their mothers. Urinary concentrations of these biomarkers reflect recent exposures (half-lives are approximately 6 to 24 hours). Biomarker concentrations in mothers' and infants' preoperative and postoperative samples were compared. RESULTS: Operations were performed in 18 infants (mean age, 5 ± 4 [SD] days). The maternal sample was obtained on postpartum day 4 ± 4. The preoperative urine sample was obtained on day-of-life 4 ± 2 and the postoperative sample on day-of-life 6 ± 4. Mean maternal concentrations for DEHP metabolites and BPA were at the 50th percentile for females in the United States general population. Infant preoperative concentrations of 1 DEHP metabolite and BPA were significantly higher than maternal concentrations. Postoperative concentrations for all DEHP metabolites were significantly greater than preoperative concentrations. CONCLUSIONS: There is considerable perioperative exposure to DEHP and BPA for neonates undergoing cardiac operations. Infant concentrations for both BPA and DEHP metabolites were significantly higher than maternal concentrations, consistent with the infant's exposure to medical devices. Further study is needed to determine the potential role of these suspect neurotoxicants in the etiology of neurodevelopmental disability after cardiac operations.

Assessment of non-derivatized ß-N-methylamino-l-alanine (BMAA) neurotoxin in free form in urine of patients with nonspecific neurological symptoms.

The beta-N-methylamino-l-alanine (BMAA) is a non-proteinogenic amino acid discussed to be produced by cyanobacteria forming harmful blooms. Since BMAA is suspected etiological agent in neurodegenerative diseases, there is a need to study and validate whether and in what concentrations can BMAA be present in human tissues. The aim of the present study was to validate analytical and extraction procedures for quantification of non-derivatized BMAA in the urine using liquid chromatography and commercial ELISA Kit. The study was focused on BMAA in different forms - dissolved, protein associated and total. The validated protocol included SPE followed by HILIC MS/MS for analyses of non-derivatized free form of BMAA with a limit of quantification 20 ng/mL. The methods for other BMAA forms (i.e. protein-associated and total) were also assessed but high matrix interferences did not allow their implementation. The method was used for analyses of free BMAA in 23 urine samples from healthy volunteers and psychiatric patients suffering from nonspecific neurological symptoms. Traces of BMAA were suspectedly detected in a single urine sample but they were not unequivocally proved according to all conservative analytical criteria. BMAA was also not confirmed in a repeatedly collected sample from the same person. The evaluated commercial BMAA ELISA Kit (Abraxis) was not suitable for determination of BMAA in extracted urine samples because of systematically highly false positive results. In agreement with recent findings, analyses of BMAA appear to methodologically challenging, and further research on BMAA in human tissues (or its precursors with potency to form BMAA under natural conditions or - eventually - during sample processing) is needed to clarify its potential ethiological role in neurodegenerative diseases.

Detection of human exposure to saxitoxin and neosaxitoxin in urine by online-solid phase extraction-liquid chromatography-tandem mass spectrometry.

Saxitoxin (STX) and neosaxitoxin (NEO) are potent neurotoxins that cause paralytic shellfish poisoning (PSP). PSP typically occurs through the ingestion of bivalve shellfish that have consumed toxin producing dinoflagellates. Due to initial presentation of symptoms being nonspecific, a clinical measurement is needed to confirm exposure to these toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of STX and NEO in human urine with tandem mass spectrometry. A unique feature of this online method is the incorporation of a new synthetic (15)N4-STX labeled internal standard used for quantitation. Manual sample preparation time was reduced by approximately 70% for 98 urine samples as compared to a previously reported method. The lowest reportable limit for STX was improved from 5.0 ng/mL to 1.01 ng/mL and from 10.0 ng/mL to 2.62 ng/mL for NEO. Three analysts validated the method with 20 calibration curves total over 30 days with precision and accuracy within ±15% for all QCs. This new online method rapidly identifies STX and NEO exposure with improved sensitivity, which can facilitate the work of public health authorities to confirm the cases of PSP, complementing the many shellfish monitoring programs worldwide.

Role of N-acetylcysteine in protecting against 2,5-hexanedione neurotoxicity in a rat model: changes in urinary pyrroles levels and motor activity performance.

The interference of N-acetylcysteine (NAC) on 2,5-hexanedione (2,5-HD) neurotoxicity was evaluated through behavioral assays and the analysis of urinary 2,5-HD, dimethylpyrrole norleucine (DMPN), and cysteine-pyrrole conjugate (DMPN NAC), by ESI-LC-MS/MS, in rats exposed to 2,5-HD and co-exposed to 2,5-HD and NAC. Wistar rats were treated with 4 doses of: 400mg 2,5-HD/kg bw (group I), 400mg 2,5-HD/kg bw+200mg NAC/kg bw (group II), 200mg NAC/kg bw (group III) and with saline (group IV). The results show a significant decrease (p<0.01) in urinary DMPN and free 2,5-HD, a significant increase (p<0.01) in DMPN NAC excretion, and a significant recovery (p<0.01) on motor activity in rats co-exposed to 2,5-HD+NAC, as compared with rats exposed to 2,5-HD alone. Taken together, our findings suggest that at the studied conditions NAC protects against 2,5-HD neurotoxicity and DMPN may be proposed as a new sensitive and specific biomarker of 2,5-HD neurotoxicity in animals treated with a toxic amount of 2,5-hexanedione.

Aluminium involvement in neurotoxicity.

The aetiology of neurodegenerative diseases (ND) seems to involve susceptibility genes and environmental factors. Toxic metals are considered major environmental pollutants. Following our study of a case of multiple sclerosis (MS) improvement due to removal of aluminium (Al) and other toxic metals, we have examined the possible relationship between Al intoxication and ND. We used the slow intravenous treatment with the chelating agent EDTA (calcium disodium ethylene diamine tetraacetic acid) (chelation test) to remove Al and detected it in the urine collected from the patients for 12 hours. Patients affected by MS represented 85.6% of total ND. Al was present in 44.8% of cases comprehensive of ND and healthy patients. Al levels were significantly higher in ND patients than in healthy subjects. We here show that treatment of patients affected by Al burden with ten EDTA chelation therapies (EDTA intravenous administration once a week) was able to significantly reduce Al intoxication.

Tissue distribution of amino acid- and lipid-brevetoxins after intravenous administration to C57BL/6 mice.

Brevetoxins produced during algal blooms of the dinoflagellate Karenia are metabolized by shellfish into reduction, oxidation, and conjugation products. Brevetoxin metabolites comprising amino acid- and lipid conjugates account for a large proportion of the toxicity associated with the consumption of toxic shellfish. However, the disposition of these brevetoxin metabolites has not been established. Using intravenous exposure to C57BL/6 mice, we investigated the disposition in the body of three radiolabeled brevetoxin metabolites. Amino acid-brevetoxin conjugates represented by S-desoxy-BTX-B2 (cysteine-BTX-B) and lipid-brevetoxin conjugates represented by N-palmitoyl-S-desoxy-BTX-B2 were compared to dihydro-BTX-B. Tissue concentration profiles were unique to each of the brevetoxin metabolites tested, with dihydro-BTX-B being widely distributed to all tissues, S-desoxy-BTX-B2 concentrated in kidney, and N-palmitoyl-S-desoxy-BTX-B2 having the highest concentrations in spleen, liver, and lung. Elimination patterns were also unique: dihydro-BTX-B had a greater fecal versus urinary elimination, whereas urine was a more important elimination route for S-desoxy-BTX-B2, and N-palmitoyl-S-desoxy-BTX-B2 persisted in tissues and was eliminated equally in both urine and feces. The structures particular to each brevetoxin metabolite resulting from the reduction, amino acid conjugation, or fatty acid addition of BTX-B were likely responsible for these tissue-specific distributions and unique elimination patterns. These observed differences provide further insight into the contribution each brevetoxin metabolite class has to the observed potencies.

Occupational exposure of air crews to tricresyl phosphate isomers and organophosphate flame retardants after fume events.

Aircraft cabin air can possibly be contaminated by tricresyl phosphates (TCP) from jet engine oils during fume events. o-TCP, a known neurotoxin, has been addressed to be an agent that might cause the symptoms reported by cabin crews after fume events. A total of 332 urine samples of pilots and cabin crew members in common passenger airplanes, who reported fume/odour during their last flight, were analysed for three isomers of tricresyl phosphate metabolites as well as dialkyl and diaryl phosphate metabolites of four flame retardants. None of the samples contained o-TCP metabolites above the limit of detection (LOD 0.5 µg/l). Only one sample contained metabolites of m- and p-tricresyl phosphates with levels near the LOD. Median metabolite levels of tributyl phosphate (TBP), tris-(2-chloroethyl) phosphate (TCEP) and triphenyl phosphate (TPP) (DBP 0.28 µg/l; BCEP 0.33 µg/l; DPP 1.1 µg/l) were found to be significantly higher than in unexposed persons from the general population. Median tris-(2-chloropropyl) phosphate (TCPP) metabolite levels were significantly not higher in air crews than in controls. Health complaints reported by air crews can hardly be addressed to o-TCP exposure in cabin air. Elevated metabolite levels for TBP, TCEP and TPP in air crews might occur due to traces of hydraulic fluid in cabin air (TBP, TPP) or due to release of commonly used flame retardants from the highly flame protected environment in the airplane. A slight occupational exposure of air crews to organophosphates was shown.

Development of a physiologically-based toxicokinetic model of acrylamide and glycidamide in rats and humans.

Physiologically-based toxicokinetic ("pharmacokinetic") (PBPK or PBTK) modeling can be used as a tool to compare internal doses of acrylamide (AA) and its metabolite glycidamide (GA) in humans and rats. An earlier PBTK model for AA and GA in rats was refined and extended to humans based on new data. With adjustments to the previous parameters, excellent fits to a majority of the data for male Fisher 344 rats were obtained. Kinetic parameters for the human model were estimated based on fit to available human data for urinary metabolites of AA, and levels of hemoglobin adducts of AA and GA measured in studies in which human volunteers ingested known doses of AA. The simulations conducted with the rat and human models predicted that rats and humans ingesting comparable levels of AA (in mg/kg day) would have similar levels of GA in blood and tissues. This finding stands in contrast to the default approach that assumes a 3.2-fold increase in human risk due to pharmacokinetic differences between rats and humans. This model was used in a companion paper to estimate safe levels of ingested AA.

Arsenic-induced neurotoxicity in relation to toxicokinetics: effects on sciatic nerve proteins.

In our previous study in rats acutely exposed to As, we observed an effect of As on neurofilaments in the sciatic nerve. This study deals with the effects of inorganic As in Wistar rats on the cytoskeletal protein composition of the sciatic nerve after subchronic intoxication. Sodium meta-arsenite (NaAsO2) dissolved in phosphate-buffered saline (PBS) was administered daily in doses of 0, 3 and 10 mg/kg body weight/day (n=9 rats/group) by intragastric route for 4, 8 and 12 week periods. Toxicokinetic measurements revealed a saturation of blood As in the 3- and 10-mg/kg dose groups at approximately 14 microg/ml, with an increase in renal clearance of As at increasing doses. After exsanguination, sciatic nerves were excised and the protein composition was analyzed. Analysis of the sciatic nerves showed compositional changes in their proteins. Protein expression of neurofilament Medium (NF-M) and High (NF-H) was unchanged. Neurofilament protein Low (NF-L) expression was reduced, while mu- and m-calpain protein expression was increased, both in a dose/time pattern. Furthermore, NF-H protein was hypophosphorylated, while NF-L and microtubule-associated protein tau (MAP-tau) proteins were (hyper)-phosphorylated. In conclusion, we show that expression of mu- and m-calpain protein is increased by exposure to As, possibly leading to increased NF-L degradation. In addition, hyperphosphorylation of NF-L and MAP-tau by As also contribute to destabilization and disruption of the cytoskeletal framework, which eventually may lead to axonal degeneration.

Biomarkers of neurotoxic shellfish poisoning.

Urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) were examined for biomarkers of brevetoxin intoxication. Brevetoxins were concentrated from urine by using solid-phase extraction (SPE), and analyzed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine extracts were fractionated by LC, and fractions analyzed for brevetoxins by ELISA. In subsequent LC-MS/MS analyses, several brevetoxin metabolites of B-type backbone were identified, with elution profiles consistent with those of ELISA. The more abundant brevetoxin metabolites in urine were characterized structurally by LC-MS/MS. With the exception of BTX-3, brevetoxin metabolites in urine differed from those found in shellfish and in shellfish meal remnants. Proposed structures of these major urinary metabolites are methylsulfoxy BTX-3, 27-epoxy BTX-3, and reduced BTX-B5. BTX-3 was found in all specimens examined. BTX-3 concentrations in urine, as determined by LC-MS/MS, correlated well with composite toxin measurements by ELISA (r(2)=0.96). BTX-3 is a useful biomarker for confirmation of clinical diagnosis of NSP.

Physiologically based modeling of n-hexane kinetics in humans following inhalation exposure at rest and under physical exertion: impact on free 2,5-hexanedione in urine and on n-hexane in alveolar air.

We used a modified physiologically based pharmacokinetic (PBPK) to describe/predict n-hexane (HEX) alveolar air concentrations and free 2,5-HD urinary concentrations in humans exposed to n-HEX by inhalation during a typical workweek. The effect of an increase in workload intensity on these two exposure indicators was assessed and, using Monte Carlo simulation, the impact of biological variability was investigated. The model predicted HEX alveolar air concentrations at rest of 19.0 ppm (25 ppm exposure) and 38.7 ppm (50 ppm exposure) at the end of the last working day (day 5), while free 2,5-HD urinary concentrations of 3.4 micromol/L (25 ppm) and 6.3 micromol/L (50 ppm) were predicted for the same period (last 4.5 hours of Day 5). Monte Carlo simulations showed that the range of values expected to occur in a group of 1000 individuals exposed to 50 ppm of HEX (95% confidence interval) for free 2,5-HD (1.7-14.7 micromol/L) is much higher compared with alveolar air HEX (33.4-46 ppm). Simulations of exposure at 50 ppm with different workloads predicted that an increase in workload intensity would not greatly affect both indicators studied. However, the alveolar air HEX concentration is more sensitive to modifications of workload intensity and time of sampling, after the end of exposure, compared with 2,5-HD. The PBPK model successfully described the HEX alveolar air concentrations and free 2,5-HD urinary concentrations measured in human volunteers and is the first, to our knowledge, to describe the excretion kinetics of free 2,5-HD in humans over a 5-day period.

High-throughput microwave-digestion procedures to monitor neurotoxic elements in body fluids by means of inductively coupled plasma mass spectrometry.

Microwave (MW) digestion procedures with high sample throughput (simultaneous digestion of 36 or 80 samples) and procedural simplicity (disposable plastic tubes, or re-usable liners with screw-cap) were investigated for their efficiency in routine analyses of biological samples. Different digestion vessel materials were tested for metal leaching/adsorption and thermal resistance: quartz, glass, polyethylene (PE) and polystyrene (PS). For the instrumental quantification of Al, Bi, Cd, Co, Cr, Hg, Mn, Mo, Ni, Pb, Sb, and Tl at ultra-trace levels in urine, serum, and whole blood, sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) was used. The different pretreatment conditions and vessels were evaluated in terms of contamination risk, effective power of detection, accuracy, and precision. Results of analyses of serum, urine and whole blood certified reference materials (CRMs) were fully satisfactory for almost all the analytes. In the case of Hg, Mo, and Tl in serum digested in plastic containers the results were just below the lower limit of uncertainty of the certified range. On the basis of the present data the following MW procedures can be suggested: 1. for urine, digestion with nitric acid at atmospheric pressure in plastic vials; 2. for serum, digestion with nitric acid at atmospheric pressure in glass vessels; and 3. for whole blood, digestion under pressure in quartz tubes. Because of the levels of the procedural blanks, Bi was not measurable at the concentrations expected in human fluids, and Al was accurately detectable in whole blood only.

Computerized posturography with sway frequency analysis: application in occupational and environmental health.

To examine the effects of occupational and environmental neurotoxicants on vestibular, cerebellar and spinocerebellar functions, the following three groups of subjects were examined, using a computerized posturography with sway frequency analysis: (1) 49 male chemical factory workers exposed to lead stearate, aged 27-63 (mean 43) years, with concurrent blood lead concentrations (BPbs) of 11-113 (mean 48) microg/100 g and past mean BPbs of 7-52 (mean 24) microg/100 g; (2) 29 male sandal, shoe and leather factory workers, aged 35-73 (mean 51) years, with urinary 2,5-hexanedione (HD) concentrations of 0.41-3.06 (mean 1.20) mg/g creatinine; and (3) 9 females, aged 19-58 (mean 29) years, who were exposed to sarin accidentally 6-8 months before the study (Tokyo Subway Sarin Poisoning, March 20,1995) and showed plasma cholinesterase (ChE) activities of 13-95 (mean 68) IU/l on the day of poisoning. The pattern of posturographic changes in lead workers suggested that the vestibulocerebellum (lower vermis), anterior cerebellar lobe and spinocerebellar afferent pathway were asymptomatically affected; the vestibulocerebellar change reflected concurrent lead absorption and the anterior cerebellar one reflected past absorption. Similarly, vestibulocerebellar and spinocerebellar functions were affected by n-hexane in solvent workers; the effect on the vestibulocerebellar function was probably inhibited by xylene. Also, the chronic (long-term) effect on the vestibulocerebellar function persisted in acute sarin poisoning. It is thus suggested that the vesitibulocerebellar function is most sensitive to all the three chemicals examined. It appears that the computerized posturography with frequency analysis is a useful technique for assessment of vestibular, cerebellar and spinocerebellar effects in occupational and environmental health.

Endogenous alkaloids in man. XXXVIII. "Chiral" and "achiral" determination of the neurotoxin TaClo (1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline) from blood and urine samples by high-performance liquid chromatography--electrospray ionization tandem mass spectrometry.

An improved sensitive assay for the determination of the dopaminergic and serotonergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) is presented, based upon on-line coupling of high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS-MS). Applying synthetic [D4]TaClo as a fourfold deuterated internal standard, TaClo was detected and reliably quantified as a trace constituent of blood samples (0.5 up to 70 ng g(-1) of clot) obtained from six patients orally treated with the hypnotic chloral hydrate. Unambiguous identification of this tricyclic "endogenous alkaloid" was achieved by selected reaction monitoring (SRM) experiments. The molecular ion peaks of TaClo, m/z 289 (for [35Cl3]TaClo) and m/z 291 (for its [37Cl35Cl2]isotopomer), were both monitored to undergo a retro-Diels-Alder fragmentation by loss of a CH2=NH portion (-29 u) as typical of a tetrahydropyrido ring system of tetrahydro-beta-carbolines. Detection of the resulting fragments, m/z 260 and m/z 262, with the expected statistical chlorine isotopic intensities of 100:96 confirmed the identity of the TaClo molecule. In addition, an enantiomer-specific device was developed for TaClo, by employing a chiral reversed-phase HPLC column in combination with circular dichroism (CD) spectroscopy and MS-MS analysis (LC-CD and LC-MS-MS coupling). In a human clot sample, both TaClo enantiomers were found in equimolar concentration (i.e., as a racemate) corroborating a spontaneous, non-enzymatic formation of TaClo from biogenic tryptamine and therapeutically administered chloral. In urine samples of TaClo-treated rats, by contrast, the (S)-antipode was found to predominate, hinting at an enantiomer-differentiating metabolism of the compound.

Interaction of zinc on biomarker responses in rats exposed to 2,5-hexanedione by two routes of exposure.

The interaction of zinc(II) on the toxicokinetics of 2,5-hexanedione (2,5-HD), the ultimate toxic metabolite of n-hexane, was performed by quantifying the changes of two urinary biomarkers, free 2,5-HD and pyrrole derivatives, in rats exposed to 2,5-HD and to 2,5-HD plus zinc acetate. Eight groups of Wistar rats were exposed for 4 days (dietary and intraperitoneally) to 2,5-HD, zinc acetate and 2,5-HD plus zinc acetate and the 24 h urine was used to determine the excretion of these biomarkers. On comparing the results obtained by the two routes of exposure with different doses of 2,5-HD and zinc acetate, it was observed that there was a significant decrease (P<0.05) in the excretion of free 2,5-HD and pyrroles derivatives in rats exposed to the chemical mixture, when compared with the excretion of these biomarkers in rats exposed to 2,5-HD alone. To evaluate the mechanism of this interaction, further experiments were performed using one group of rat dietary pre-exposed to zinc acetate followed by 2,5-HD exposure. The results of our experiment suggest that zinc protect proteins of pyrrolization by coordination to amino groups, with the subsequent inhibition of protein cross-linking responsible by 2,5-HD neurotoxicity.

Relationship between 2,5-hexanedione concentrations in nerve, serum, and urine alone or under co-treatment with different doses of methyl ethyl ketone, acetone, and toluene.

To ascertain the relationship among 2,5-hexanedione (2,5-HD) concentrations in nerve, serum and urine, rats were injected subcutaneously with 2.6 mmol/kg 2,5-HD alone, or together with 2.6 or 13.0 mmol/kg of methyl ethyl ketone, acetone and toluene. 2,5-HD concentrations in sciatic nerve (NC), serum (SC) and urine (UC) were determined, and the linear regression between each two of NC, SC, and UC were calculated. There was good correlation between NC and SC, SC and UC in the 2,5-HD alone group, and good correlation between NC and SC in the co-treated groups. Co-treatment solvent had little effect on the relationship between SC and NC. 13.0 mmol/kg co-treated solvent tended to decrease the regression coefficients compared with 2.6 mmol/kg co-treated solvent. These results show that SC can be used in estimating NC in the 2,5-HD alone or co-treated groups, and UC can be used in estimating SC in the 2,5-HD alone group.

Urinary eosinophil-derived neurotoxin/protein X: a simple method for assessing eosinophil degranulation in vivo.

BACKGROUND: Eosinophil-derived neurotoxin/protein X (EDN/EPX), one of the cationic granule proteins released by polymorphonuclear eosinophils, can be detected in human urine. OBJECTIVE: We sought to evaluate whether the urinary release of EDN/EPX was dependent on the blood eosinophil cell count, the bronchoalveolar eosinophil cell count, or both and on the clinical diagnosis. We also attempted to determine the precise kinetics of decrease of EDN excretion and eosinophil counts after the onset of corticosteroid treatment. METHODS: Daily urinary release of EDN/EPX was measured by radioimmunoassay in 28 patients with high hypereosinophilia (group 1), 32 patients with moderate hypereosinophilia (group 2), 26 patients without hypereosinophilia at the time of the study but with a known pulmonary disease involving eosinophils (group 3), and 13 control patients (group 4). RESULTS: The urinary excretion of EDN/EPX was significantly higher in patients from groups 1 or 2 than in patients from groups 3 or 4. Particularly high levels of EDN/EPX excretion were observed in patients from groups 1 or 2 with chronic eosinophilic pneumonia (chronic eosinophilic pneumonia: 4.7 +/- 8.1 mg/day, control subjects: 0.39 +/- 0.33 mg/day, p < 0.001). Urinary excretion of EDN/EPX was significantly correlated with blood (r = 0.66, p < 0.001) and differential bronchoalveolar (r = 0.62, p = 0.04) eosinophil cell counts in patients from group 1 but not from the other groups. Corticosteroid treatment was followed by a significant decrease in EDN/EPX excretion. The kinetics of decrease in EDN/EPX were delayed as compared with the dramatic drop in peripheral eosinophil counts. Distinct kinetics between urinary EDN/EPX and eosinophil counts differentiated the recurrence of chronic eosinophilic pneumonia from an asthma attack in one patient. CONCLUSION: Measurement of urinary EDN/EPX excretion may be a useful indicator of eosinophil degranulation in vivo.