Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Mycobacterium species, Nocardia species, and Other Aerobic Actinomycetes.

The value of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed.

Effect of ascorbic acid dose taken with a meal on nitrosoproline excretion in subjects ingesting nitrate and proline.

We determined the dose of ascorbic acid (ASC) given to subjects with a standard 400-calorie meal that inhibited N-nitrosoproline (NPRO) formation when we gave 400 mg of nitrate one hour before and 500 mg of L-proline with the standard meal. Volunteers consumed their normal US diets but restricted their intakes of nitrate, proline, NPRO, and ASC. NPRO and N-nitrososarcosine (NSAR) were determined in the 18-hour urines by methylation followed by gas chromatography-thermal energy analysis. Mean NPRO yields were 10.7, 41.9, 33.2, 22.3, and 23.1 nmol for groups of 9-25 subjects taking proline alone, proline + nitrate, and proline + nitrate + 120, 240, and 480 mg of ASC, respectively. There was a significant trend to lower NPRO yields as the ASC dose was raised. These results correspond to inhibitions by ASC of 28%, 62%, and 60%, respectively. Pairwise comparison showed that each group taking ASC formed significantly less NPRO than the group given only proline + nitrate. Mean NSAR yields were 9.0 nmol when proline alone was taken and 16.9-24.0 nmol when proline + nitrate + ASC was taken, with no trend to increase as the ASC dose was raised. However, NPRO and NSAR yields in individual urines were correlated with each other. We concluded that 120 mg of ASC taken with each meal (360 mg/day) would significantly reduce in vivo nitrosamine formation, similar to tests by Leaf and co-workers (Carcinogenesis 8, 791-795, 1987) in which the reactants were taken between meals. The inhibitory dose of ASC may be < 120 mg/meal when doses of nitrate and proline are not taken.

Dosing time with ascorbic acid and nitrate, gum and tobacco chewing, fasting, and other factors affecting N-nitrosoproline formation in healthy subjects taking proline with a standard meal.

The N-nitrosoproline (NPRO) test measures the potential for intragastric formation of carcinogenic nitrosamines in humans. Nitrate and L-proline are administered to volunteers. Noncarcinogenic NPRO is produced by an acid-catalyzed reaction of proline (a model for ingested amines) with nitrate-derived nitrite in the stomach. It is then absorbed and excreted in the urine, which is analyzed for NPRO. We studied the effect of certain dietary and other factors on the levels of urinary NPRO. For (generally) 5 days, healthy adult subjects (mostly men) followed a diet low in preformed NPRO, nitrate, proline, and (on days 4 and 5) ascorbic acid. The tests were conducted on days 4 and 5. In the standard test, the subjects took 400 mg nitrate at 11 a.m., and at noon they ate a standard 700-calorie meal containing 500 mg proline. (In previous tests, proline was given 1 h after or between meals.) Urines were collected for 24 h, and samples were analyzed for NPRO by published methods. This standard test yielded 26 +/- 2 (mean +/- SE) nmol NPRO compared with 5 +/- 1 nmol NPRO when proline alone was taken. In variations of the standard test, NPRO yield was not significantly affected by the subjects' gender, the time at which the standard meal was eaten, the size of the meal, or the drinking of extra water after the meal. Doses of 100 and 200 mg nitrate had lesser effects on NPRO yield than did the dose of 400 mg nitrate. Nitrate (400 mg) produced the most NPRO when it was given 1 h before the meal. Fasting increased NPRO yield by 3-4 times compared to giving proline with a meal. One g of ASC given 5 or 2 h before, with, or 1 or 2 h after the meal with proline inhibited NPRO formation by mean values of 0, 71, 71, 67, and 19%, respectively. Chewing gum or tobacco for 2-3 h after the test meal did not increase NPRO formation or salivary nitrate levels, but salivary nitrite was not taken, chewing tobacco appeared to increase salivary nitrite and nitrate levels. The weak carcinogen N-nitrososarcosine (NSAR) was also detected in some tests, and the standard group showed 21 +/- 3 nmol NSAR. A high NSAR result (44 +/- 7 nmol) for women undergoing the standard test should be reexamined. We discuss applying these results to the conduct of future NPRO tests, as well as their implications for reducing the potential production of carcinogenic nitrosamines in the stomach.

Vagal cholinergic innervation of the airways in newborn cat and dog.

Although several studies have examined the pulmonary response to muscarinic agonists in the newborn, none has addressed the functional capabilities or "maturity" of vagal innervation to airway smooth muscle in the newborn. The purpose of the present study was to provide a quantitative analysis of the ability of vagal excitatory innervation (encompassing the pre- and postganglionic fibers, airway ganglia, and airway smooth muscle) to alter pulmonary mechanics in the newborn. We measured the changes in pulmonary mechanics elicited by electrical stimulation of the vagus nerves in 20 newborn cats and 18 puppies anesthetized with chloralose urethan. Animals were tracheotomized and ventilated (chest open), and the cervical vagus nerves were sectioned and placed on stimulating electrodes. Animals were placed in a flow plethysmograph, and mean inspiratory resistance (RL,I) and dynamic compliance were measured on a breath-by-breath basis. In each animal RL,I increased, dynamic compliance decreased, and heart rate slowed during 10 s of vagal stimulation at frequencies ranging from 2 to 20 pulses/s. At each stimulus frequency there was a spectrum of responses with respect to the percent change in RL,I. At 15 pulses/s there was a fourfold difference in the RL,I response of the most- and least-sensitive animals. In both species, higher stimulus frequencies caused greater increases in RL,I; at 2 pulses/s RL,I increased on average approximately 40%, compared with approximately 250% at 20 pulses/s. The increase in RL,I was similar in the kitten and puppy at stimulus frequencies of 6 and 15 pulses/s but was less in the kitten at 2 pulses/s (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Nonadrenergic inhibitory innervation to the airways of the newborn cat.

Vagal, nonadrenergic inhibitory system (NAIS) innervation to airway smooth muscle has been demonstrated in adults of several species, including humans. However, the functional status of this system in newborns is not known. The NAIS of intestinal smooth muscle has been demonstrated in newborns and develops in parallel with cholinergic innervation (14). Since the lung is derived embryologically from the foregut and cholinergic innervation is operative at birth, we tested the hypothesis that NAIS innervation to the airways is functional in newborn cats. Nineteen cats (2-11 days of age) were anesthetized with chloralose-urethan, and a tracheal cannula was inserted. The chest was opened and the animals were mechanically ventilated. The cervical vagus nerves were separated from the sympathetics, cut, and placed on stimulating electrodes. Mean inspiratory resistance (RL, I) and dynamic compliance (Cdyn, L) were measured on a breath-by-breath basis. Atropine and propranolol were administered (2 mg/kg iv) to block cholinergic and adrenergic pathways, respectively. Subsequently, serotonin infusion was used to increase RL, I approximately 150%. Stimulation (10 s) at frequencies ranging from 2 to 20/s caused a slow-onset (30 s to peak) long-lasting decrease in RL, I and a much smaller increase in Cdyn, L. The magnitude and duration of the bronchodilation increased with stimulus frequency to a plateau at approximately 15/s. At a stimulus frequency of 2/s, RL, I decreased 11 +/- 1.9 vs 36 +/- 4.8% (SE) at 20/s, whereas Cdyn, L increased 2 +/- 1.1 vs. 6 +/- 1.7%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)