Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents.

Epidemiologic reports by C.A. Pope III et. al. demonstrated that in the Utah Valley, closure of an open-hearth steel mill over the winter of 1987 was associated with reductions in respiratory disease and related hospital admissions in valley residents. To better examine the relationship between plant-associated changes in ambient particulate matter (PM) and respiratory health effects, we obtained total suspended particulate filters originally collected near the steel mill during the winter of 1986 (before closure), 1987 (during closure), and 1988 (after plant reopening). PM subcomponents were water-extracted from these filters and Sprague-Dawley rats were intratracheally instilled with equivalent masses of extract. Data indicated that 24 hr later, rats exposed to 1986 or 1988 extracts developed significant pulmonary injury and neutrophilic inflammation. Additionally, 50% of rats exposed to 1986 or 1988 extracts had increased airway responsiveness to acetylcholine, compared to 17 and 25% of rats exposed to saline or the 1987 extract, respectively. By 96 hr, these effects were largely resolved except for increases in lung lavage fluid neutrophils and lymphocytes in 1986 extract-exposed rats. Analogous effects were observed with lung histologic assessment. Extract analysis using inductively coupled plasma-mass spectroscopy demonstrated in all three extracts nearly 70% of the mass appeared to be sodium-based salts derived from the glass filter matrix. Interestingly, relative to the 1987 extract, the 1986/1988 extracts contained more sulfate, cationic salts (i.e., calcium, potassium, magnesium), and certain metals (i.e., copper, zinc, iron, lead, strontium, arsenic, manganese, nickel). Although total metal content was (3/4) 1% of the extracts by mass, the greater quantity detected in the 1986 and 1988 extracts suggests metals may be important determinants of the pulmonary toxicity observed. In conclusion, the pulmonary effects induced by exposure of rats to water-based extracts of local ambient PM filters were in good accord with the cross-sectional epidemiologic reports of adverse respiratory health effects in Utah Valley residents.

Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism.

Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 microg/cm(2) ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.

Ozone effects on airway responsiveness, lung injury, and inflammation. Comparative rat strain and in vivo/in vitro investigations.

Asthmatic individuals appear to be particularly sensitive to the effects of certain air pollutants-including ozone (O(3)), an oxidant ambient air pollutant-for reasons that are poorly understood. The general purpose of these studies, therefore, was to expand and improve upon toxicologic methods for assessing ozone-induced effects on the airways of the rat by (1) developing an in vivo testing procedure that allows detection of airway responsiveness changes in rats exposed to ozone; (2) identifying a strain of rat that may be inherently more sensitive to the effects of ozone; and (3) validation of an in vitro epithelial culture system to more directly assess airway cellular/subcellular effects of ozone. Using methacholine inhalation challenges, we detected increased airway responsiveness in senescent F344 rats acutely after ozone exposure (2 ppm x 2 h). We also determined that acutely after ozone exposure (0.5 ppm x 8 h), Wistar rats developed significantly greater lung injury, neutrophilic inflammation, and bronchoalveolar lavage (BAL) fluid concentrations of IL-6 than either Sprague-Dawley (SD) or F344 rats. SD rats had greater BAL fluid concentrations of prostaglandin E(2) (PGE(2)), while F344 rats consistently exhibited the least effect. Wistar rat-derived tracheal epithelial (RTE) cultures were exposed in vitro to air or ozone (0.1-1.0 ppm x 1 h), and examined for analogous effects. In a concentration-dependent manner, ozone exposure resulted in acute but minor cytotoxicity. RT polymerase chain reaction (PCR) analysis of RNA isolated from ozone-exposed cells demonstrated variable increases in steady-state gene expression of IL-6 at 4 h postexposure, while at 24 h cellular fibronectin expression (EIIIA domain) was decreased. Exposure was without effect on macrophage inflammatory protein 2 (MIP-2) or gamma-glutamyl cysteine synthetase expression. At 6 h postexposure, IL-6 synthesis and apical release appeared increased in ozone-exposed cells (1 ppm x 1 h). MIP-2 release was not significantly increased in ozone-exposed cells. At 2 h postexposure, ozone exposure resulted in minor increases in apical fibronectin, but exposure was without effect on basolateral accumulation of fibronectin. Exposure to 1.0, but not 0.1 ppm (x 1 h), increased production of cyclooxygenase (i.e., PGE(2)) and noncyclooxygenase products of arachidonic acid. Results demonstrate that multiple inflammatory mediator pathways are affected by ozone exposure. Such effects could exacerbate morbidity in individuals with preexisting airway inflammation such as asthmatics.

Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression.

Particulate matter (PM) metal content and bioavailability have been hypothesized to play a role in the health effects epidemiologically associated with PM exposure, in particular that associated with emission source PM. Using rat tracheal epithelial cells in primary culture, the present study compared and contrasted the acute airway epithelial effects of an emission source particle, residual oil fly ash (ROFA), with that of its principal constitutive transition metals, namely iron, nickel, and vanadium. Over a 24-h period, exposure to ROFA, vanadium, or nickel plus vanadium, but not to iron or nickel, resulted in increased epithelial permeability, decreased cellular glutathione, cell detachment, and lytic cell injury. Treatment of vanadium-exposed cells with buthionine sulfoximine further increased cytotoxicity. Conversely, treatment with the radical scavenger dimethylthiourea inhibited the effects in a dose-dependent manner. RT-PCR analysis of RNA isolated from ROFA-exposed rat tracheal epithelial cells demonstrated significant macrophage inflammatory protein-2 and interleukin-6 gene expression as early as 6 h after exposure, whereas gene expression of inducible nitric oxide synthase was maximally increased 24 h postexposure. Again, vanadium (not nickel) appeared to be mediating the effects of ROFA on gene expression. Treatment with dimethylthiourea inhibited both ROFA- and vanadium-induced gene expression in a dose-dependent manner. Corresponding effects were observed in interleukin-6 and macrophage inflammatory protein-2 synthesis. In summary, generation of an oxidative stress was critical to induction of the ROFA- or vanadium-induced effects on airway epithelial gene expression, cytokine production, and cytotoxicity.

The role of reactive oxygen and nitrogen species in the response of airway epithelium to particulates.

Epidemiologic and occupational studies indicate adverse health effects due to inhalation of particulate air pollutants, but precise biologic mechanisms responsible have yet to be fully established. The tracheobronchial epithelium forms the body's first physiologic barrier to such airborne pollutants, where ciliary movement functions to remove the offending substances caught in the overlying mucus layer. Resident and infiltrating phagocytic cells also function in this removal process. In this paper, we examine the role of reactive oxygen and nitrogen species (ROS/RNS) in the response of airway epithelium to particulates. Some particulates themselves can generate ROS, as can the epithelial cells, in response to appropriate stimulation. In addition, resident macrophages in the airways and the alveolar spaces can release ROS/RNS after phagocytosis of inhaled particles. These macrophages also release large amounts of tumor necrosis factor alpha (TNF-alpha), a cytokine that can generate responses within the airway epithelium dependent upon intracellular generation of ROS/RNS. As a result, signal transduction pathways are set in motion that may contribute to inflammation and other pathobiology in the airway. Such effects include increased expression of intercellular adhesion molecule 1, interleukin-6, cytosolic and inducible nitric oxide synthase, manganese superoxide dismutase, cytosolic phospholipase A2, and hypersecretion of mucus. Ultimately, ROS/RNS may play a role in the global response of the airway epithelium to particulate pollutants via activation of kinases and transcription factors common to many response genes. Thus, defense mechanisms involved in responding to offending particulates may result in a complex cascade of events that can contribute to airway pathology.

Epithelial injury induced by exposure to residual oil fly-ash particles: role of reactive oxygen species?

Exposure of animals to airborne particulates is associated with pulmonary injury and inflammation. In the studies described here, primary cultures of rat tracheal epithelial (RTE) cells were exposed to suspensions of residual oil fly ash (ROFA). ROFA exposure resulted in progressive cytotoxicity whereby the amount of lactate dehydrogenase (LDH) released was significantly greater at 24 h than at 6 h after exposure. In a dose-dependent manner, exposure to 5, 10, or 20 microg/cm2 of ROFA for 24 h resulted in cytotoxicity and detachment of cells from the collagen matrix, along with altered permeability of the RTE cell layer. ROFA exposure caused cellular glutathione levels to decrease, producing a condition of oxidative stress in the RTE cells. Treatment of RTE cells with buthionine sulfoxamine, an inhibitor of gamma-glutamyl cysteine synthetase, was found to augment ROFA-induced cytotoxicity. Treatment with dimethylthiourea (DMTU) inhibited ROFA-induced LDH release and permeability changes in a dose-dependent manner. Treatment with the nitric oxide synthase inhibitor NG-monomethyl-D-arginine (D-NMA) for 24 h was without effect. In rats intratracheally instilled with ROFA (500 microg/rat), intraperitoneal administration of DMTU (500 mg/kg) significantly ameliorated the degree of pulmonary neutrophilic inflammation present at 24 h. Overall, these in vitro findings suggest that ROFA-induced RTE cell injury may be mediated by hydroxyl-radical-like reactive oxygen species (i.e., species scavenged by DMTU) that are generated via non-nitric oxide pathways. The delay in induction of maximal RTE cell injury may reflect the time necessary to produce an oxidative burden by depleting antioxidant defenses such as cellular glutathione.

Bronchopulmonary disease in the cat: historical, physical, radiographic, clinicopathologic, and pulmonary functional evaluation of 24 affected and 15 healthy cats.

The results of clinical and pulmonary functional evaluation of 24 cats with bronchopulmonary disease and 15 healthy cats are presented. Affected cats had historical evidence of excessive reflexes (coughing, sneezing); physical evidence of airway secretions (crackles), obstruction (wheezing), and increased tracheal sensitivity; radiographic evidence of bronchial and interstitial lung disease; and cytological evidence of airway inflammation or mucous secretions. Bacterial isolates from healthy and affected cats were predominantly Gram-negative rods, indicating that bronchi of cats are not always sterile and that normal flora should be considered in interpreting cultures from cats with suspected bronchopulmonary disease. Cats were grouped according to relative disease severity based on scored historical, physical, and radiographic abnormalities. The mean (+/- standard deviation) baseline lung resistance measurement in healthy cats was 28.9 cm H2O/L/s (+/- 6.2 cm H2O/L/s), whereas in mildly, moderately, and severely affected cats it was 38.3 cm H2O/L/s (+/- 21.5 cm H2O/L/s), 44.8 cmH2O/L/s (+/- 7.7 cm H2O/L/s), and 105.2 cm H2O/L/s (+/- 66.9 cm H2O/L/s), respectively. In healthy cats, dynamic lung compliance was 19.8 (+/- 7.4), whereas in mildly, moderately, and severely affected cats it was 14.7 mL/cm H2O (+/- 3.8 mL/cm H2O), 17.7 mL/cm H2O (+/- 6.9 mL/cm H2O), and 13.0 mL/cm H2O (+/- 7.9 mL/cm H2O), respectively. Thus, airway obstruction was present in many of the affected cats. Based on acute response to the bronchodilator, terbutaline, airway obstruction was partially reversible in many affected cats, although the degree of reversibility varied. Furthermore, based on bronchoprovocation testing, 6 (of 7) affected cats evaluated also had increased airway responsiveness to aerosolized methacholine.

Characterization of upper respiratory disease in rats following neonatal inoculation with a rat-adapted influenza virus.

Neonatal F344 rats were infected with a rat-adapted influenza virus (RAIV) to use as a potential model to study the combined effects of air pollutant exposure with early life respiratory viral infections. Initially, 6-day-old pups were intranasally inoculated with RAIV or medium alone, and nasal and lower respiratory tract (LRT) tissues were assessed histologically at 1, 3, 6, and 13 days postinoculation (DPI). Immunologic assessments included thymic lymphocyte quantification and anti-RAIV immunoglobulin production. Pups then received two inoculations (at 6 and 30 days of age), with histologic and immunologic assessment 6 and 13 days after the second inoculation and bronchoprovocation testing 5-8 weeks later. Following the single RAIV inoculation, IgM and IgG1 measurements increased at 6, 11, and 15 DPI, with IgG1 being greater at 11 and 15 DPI. Nasal lesions were evident as early as 1 DPI and primarily involved the anterior dorsal medial meatus and adjacent dorsal atrio- and nasoturbinates. Alterations included epithelial cell exfoliation and necrosis, mild erosions, suppurative and nonsuppurative inflammation, intraepithelial neutrophil accumulations, and intraluminal exudate. By 3 DPI, olfactory epithelial damage was multifocal or locally diffuse, with degeneration of sensory cells and variable inflammation. By 13 DPI, lesions were essentially repaired. Minimal changes were apparent in the LRT despite evidence of viral replication in the lungs 24 hours after inoculation (> 3 log10 plaque-forming units/lung). Pups reinoculated with RAIV at 30 days of age did not develop significant histologic lesions, nor did they exhibit increased airway responsiveness when assessed as young adults. In spite of their immature immune status at the time of initial infection, 13 days after the second RAIV inoculation, IgG1 increased substantially. Thus, neonatal RAIV infection resulted in acute nasal epithelial injury and inflammation, alterations that may allow subsequent evaluation of viral disease-air pollutant interactions.

Ocular and disseminated candidiasis in an immunosuppressed cat.

Ocular and systemic candidiasis was diagnosed in an immunosuppressed and diabetic 12-year-old cat that initially was examined because of polyuria, polydipsia, and urinary tract disease. Bilateral recurrent corneal erosions and chorioretinitis, urinary tract infections attributable to bacteria or Candida sp, and renal dysfunction developed during the next 2 months. Examination of corneal scrapings revealed spherical to oval, budding, yeast-like cells. The cat's condition progressively deteriorated, and it was euthanatized. Toxoplasmosis was diagnosed by fecal flotation and from serum titers, and pituitary-dependent hyperadrenocorticism was detected at postmortem histologic evaluation. Candida budding yeasts and pseudohyphae with blastospores were detected in the corneas, vitreous bodies, retinas, CNS, pharynx, trachea, esophagus, kidneys, and urinary bladder at postmortem examination.

Tidal breathing flow-volume loops in healthy and bronchitic cats.

Tidal breathing flow-volume loops (TBFVL) were obtained from 19 healthy cats and 7 cats with chronic bronchial disease. Peak inspiratory flow (PIF) occurred late in the inspiratory cycle and was preceded by a gradual but more linear increase in the flow rate. Peak expiratory flow (PEF) occurred early during expiration and was followed by a curvilinear decrease in flow to a point near the end of expiration where flow ceased. The loops obtained were generally reproducible. The mean coefficient of variation (CV) for TBFVL indices of healthy cats ranged from 5.6% to 21.9%. Loop indices from cats with chronic bronchial disease had a mean CV between 6.6% and 28.4%. Significant differences were noted in the bronchitic cats' TBFVLs, including an increased ratio of expiratory time to inspiratory time, lower expiratory flow rates, decreased area under total and peak expiratory flow curves, and decreased tidal breathing expiratory volumes (TBEV) at 0.1 and 0.5 seconds. Selected TBFVL indices were also significantly reduced. TBFVL evaluation in the cat is easy to perform, is reproducible, and has allowed for the detection of changes during tidal breathing in cats with histories and physical findings of chronic lower airway disease.

Acute methanol toxicity in minipigs.

The pig has been proposed as a potential animal model for methanol-induced neuro-ocular toxicosis in humans because of its low liver tetrahydrofolate levels and slower rate of formate metabolism compared to those of humans. To examine the validity of this animal model, 12 4-month-old female minipigs (minipig YU) were given a single oral dose of water or methanol at 1.0, 2.5, or 5.0 g/kg body wt by gavage (n = 3 pigs/dose). Dose-dependent signs of acute methanol intoxication, which included mild CNS depression, tremors, ataxia, and recumbency, developed within 0.5 to 2.0 hr, and resolved by 52 hr. Average maximum methanol concentrations in plasma, of 3100 +/- 700 (SD), 6200 +/- 2300, and 15,200 +/- 900 micrograms/ml were reached within 0.5 to 4 hr following methanol administration in animals given 1.0, 2.5, or 5.0 g methanol/kg, respectively. The mean initial elimination half-lives of methanol were 9.0 +/- 1.6, 22.4 +/- 6.1, and 18.9 +/- 4.3 hr, for 1, 2.5, and 5.0 g/kg doses, respectively. In 3 minipigs, a transient increase in plasma formate concentration (1.74-3.40 mEq/liter vs control = 0.5 +/- 0.3 mEq/liter) occurred 4 to 30 hr following methanol administration. Methanol- and formate-dosed pigs did not develop optic nerve lesions, toxicologically significant formate accumulation, or metabolic acidosis. Based on results following a single dose, female minipigs do not appear to be overtly sensitive to methanol and thus may not be a suitable animal model for acute methanol-induced neuro-ocular toxicosis.

Feline bronchopulmonary disease.

This author is aware that not all cats fit so neatly into these subcategories. It is hoped, however, that through increased awareness of the differences between cats with bronchopulmonary disease, we can begin to focus and refine our diagnostic and therapeutic efforts and more accurately predict the prognosis of individual cats. The pulmonary functional changes in the cats presented here lend credence to the clinical use of these airway disease subtypes. However, relative to human disease syndromes, our current understanding of feline bronchopulmonary disease is in its infancy. One can only speculate as to why these cats developed airway inflammation in the first place. Constant exposure to dust through litter use or upper respiratory tract infections, seem to be likely causes. But, why do only certain cats become clinically ill when all cats are potentially exposed to similar conditions? Owing to the diversity of disease present in these cats, it is likely that multiple etiologies are involved. Alpha 1-antiprotease deficiency, for example, is a known genetic defect associated with the development of panacinar emphysema in homozygous humans. It is conceivable that similar genetic defects could be present in individual cats of the Siamese breed, as this breed is overrepresented in this syndrome and some of these cats appear to follow a progressive disease course.

Chronopharmacokinetics of theophylline in the cat.

Studies of theophylline pharmacokinetics in humans have shown that a higher peak concentration and area under the curve (AUC), with a shorter time to peak (tp) occur after a morning dose than after an evening dose. The purpose of this study was to determine whether theophylline pharmacokinetics in the cat were also influenced by the administration time of day. Theophylline was administered to six cats in a three-way cross-over study as a single dose of intravenous aminophylline and oral sustained-release theophylline (Slo-bid Gyrocaps and Theo-Dur Tablets), between 08.00-09.00 h (Phase I) and 20.00-21.00 h (Phase II). Subjects were maintained on a 12-h light (08.00-20.00 h): 12-h dark cycle. Similar to the human studies, the tp was shorter following the morning dose. Conversely, however, the peak plasma theophylline concentrations achieved in these cats following intravenous aminophylline and oral Slo-bid were significantly higher following the evening dose. The AUC obtained for Theo-Dur was also significantly greater following the evening dose. No single pharmacokinetic parameter could account for the higher plasma concentrations achieved following the evening dose.

Sustained-release theophylline pharmacokinetics in the cat.

Theophylline was administered in a three-way crossover design study to six cats intravenously (Aminophylline USP, Invenex Laboratories, Chagrin Falls, OH) and orally as two sustained-release formulations (Slo-bid Gyrocaps (SB), William H. Rorer, Inc., Fort Washington, PA; Theo-Dur Tablets (TD), Key Pharmaceuticals, Miami, FL). Values were determined for mean residence time (SB = 19.4 +/- 3.2 h; TD = 15.8 +/- 4.8 h), mean absorption time (SB = 8.0 +/- 2.3 h; TD = 4.8 +/- 2.3 h), absolute bioavailability (SB = 82 +/- 27%; TD = 76 +/- 38%), and time to peak plasma concentrations (SB = 8 h; TD = 8 h). After normalization to a dose of 25 mg/kg, the average peak plasma concentrations were also predicted (SB = 10.5 +/- 3.4 micrograms/ml; TD = 14.3 +/- 6.7 micrograms/ml). Slo-bid was predicted to provide the least peak:trough fluctuation in theophylline concentrations. Slo-bid and Theo-Dur appear to have pharmacokinetic characteristics which, if given once-daily, would maintain plasma theophylline concentrations of 5-20 micrograms/ml in the cat.

Effect of alcohols and selected solvents on serum osmolality measurements.

The method by which serum osmolality is measured can significantly affect the result if certain volatiles or solvents are present in the specimen. Commonly available solvents and alcohols were added to aliquots of pooled human serum to produce toxicologically relevant concentrations. Increasing concentrations of carbon tetrachloride, chloroform, mono-n-butyl ether (butyl cellosolve), 1, 1,1 trichloroethylene, toluene, and xylene did not change vapor pressure (VP) or freezing point depression (FPD) osmolality. Acetone, ethanol, isopropanol, and methanol in increasing concentrations produced a linear increase in FPD osmolality, but no change in VP osmolality. Only ethylene glycol produced a linear increase in VP and FPD osmolality across the range of concentrations studied. Despite the excellent correlation between osmolality and ethanol concentration in prepared serum samples, this relationship could not accurately predict patient ethanol concentrations from FPD osmolality. The osmolal gap, "delta" osmolality, (measured FPD minus calculated osmolality) did not correlate with the difference between measured FPD and VP osmolalities. Patient ethanol levels could not be predicted with accuracy using an equation based on the osmolal gap or "delta" osmolality.

Rate-nephelometric inhibition immunoassay of phenytoin and phenobarbital.

A proposed rate-nephelometric inhibition immunoassay of phenytoin and phenobarbital in human serum involves the sequential addition to buffer of 42-microL aliquots of sample containing the hapten (drug) of interest, a hapten conjugate (drug-equine apoferritin), and specific antibody to the hapten. The drug and the drug conjugate compete for the binding sites on the antibody. The free hapten-antibody complex is soluble and so does not scatter light, whereas the complex of antibody and drug conjugate is insoluble and thus scatters light. The latter immunoprecipitation is competitively inhibited by free hapten. Thus the higher the concentration of free hapten present, the fewer immunoprecipitin complexes are formed, and the less light scatter. Precision, accuracy, linearity, analytical recovery, and comparison with patients' samples assayed with the DuPont aca were excellent. There was no significant interference from hemolysis, icterus, or lipemia. Many potentially interfering drugs and metabolites were checked for cross reactivity, with negative results. Reaction times range from 30 to 50 s.

A modified ion-selective electrode method for measurement of chloride in sweat.

A modified method of analysis of sweat chloride concentration with an ion-selective electrode is presented. The original method of sweat chloride analysis proposed by the Orion Research Corporation (Cambridge, Massachusetts 02139) is inadequate because it produces erratic and misleading results. The modified method was compared with the reference quantitative method of Gibson and Cooke. In the modified method, individual electrode pads are cut and placed in the electrodes rather than using the pads supplied by the company; pilocarpine nitrate (2,000 mg/l) is used in place of pilocarpine HCl (640 mg/l); sodium bicarbonate as the weak electrolyte is used instead of K2SO4. A 10-minute period for sweat accumulation is employed rather than a zero-time collection as in the original Orion method. The modification has been studied for reproducibility in individuals, reproducibility between right and left arm in individuals; it has been compared extensively with the quantitative method of Gibson and Cooke, both in normal individuals and in patients with cystic fibrosis. There is excellent agreement between the modified method and the quantitative reference method. There appears to be a slight bias toward higher concentrations of chloride from the right arm compared with the left arm, but this difference is not medically significant.