Site of airway obstruction in pulmonary disease: direct measurement of intrabronchial pressure.

To partition the central and peripheral airway resistance in awake humans, a catheter-tipped micromanometer sensing lateral pressure of the airway was wedged into the right lower lobe of a 3-mm-ID bronchus in 5 normal subjects, 7 patients with chronic bronchitis, 8 patients with emphysema, and 20 patients with bronchial asthma. We simultaneously measured mouth flow, transpulmonary pressure, and intra-airway lateral pressure during quiet tidal breathing. Total pulmonary resistance (RL) was calculated from transpulmonary pressure and mouth flow and central airway resistance (Rc) from intra-airway lateral pressure and mouth flow. Peripheral airway resistance (Rp) was obtained by the subtraction of Rc from RL. The technique permitted identification of the site of airway resistance changes. In normal subjects, RL was 3.2 +/- 0.2 (SE) cmH2O.l-1.s and the ratio of Rp to RL was 0.24 during inspiration. Patients with bronchial asthma without airflow obstruction showed values of Rc and Rp similar to those of normal subjects. Although Rc showed a tendency to increase, only Rp significantly increased in those patients with bronchial asthma with airflow obstruction and patients with chronic bronchitis and emphysema. The ratio of Rp to RL significantly increased in three groups of patients with airflow obstruction (P less than 0.01). These observations suggest that peripheral airways are the predominant site of airflow obstruction, irrespective of the different pathogenesis of chronic airflow obstruction.     

A bioluminescence method for direct measurement of phosphodiesterase activity

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25–10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

A technique for quantitating airway size from bronchial casts

To develop a technique for quantitating the size of airways at various positions in the bronchial tree, we analyzed casts of formalin-fixed excised lungs of five mature male ferrets. The left lower lobe of each cast was dissected, the diameter and position of each terminal bronchiole were entered into a computer programmed to reconstruct the airway system, and the cross-sectional areas of 120 conducting airways were measured. The fraction of the lobe served by each measured airway was estimated by dividing the sum of the squared diameters of the terminal bronchioles subtended by that airway by the summed squared diameters of all terminal bronchioles in the lobe. In each cast the relationship between an airway's cross-sectional area (Y) and the fraction of the lobe it was estimated to subtend (X) was described (0.91 less than R2 less than 0.95) by the expression ln(Y) = A + B ln(X) + C [ln(X)]2. Linear regression of ln(Y) on ln(X) for 30-50 airways estimated to serve fractions of the lobe around each of three arbitrarily selected levels (airways serving 0.7, 2.2, and 9.5% of the lobe) was adequate to characterize the area of airways at each level in each of the five animals with 95% confidence intervals narrower than 8% of the estimated area. Variability of airway size at each level among the five casts was modest, suggesting that this technique identified analagous airways in the various animals. Interindividual variability did not increase when the data were reanalyzed with terminal units defined on the basis of airway diameters rather than on the morphological identification of terminal bronchioles.     

A novel technique for measurement of pericardial pressure

To determine whether pericardial liquid pressure accurately measures pericardial constraint, we developed a technique in which a catheter was positioned perpendicular to the epicardial surface. This device, which occupies little or no pericardial space, couples the thin film of liquid to a transducer. In six open-chest dogs, we also measured left ventricular (LV) end-diastolic pressure (LVEDP) and anteroposterior and septum-to-free wall diameters. LVEDP was raised incrementally to approximately 25 mmHg by saline infusion. With the use of the product of the two diameters as an index of area (A(LV)), LVEDP-A(LV) relationships were obtained with the pericardium closed and again after the pericardium had been widely opened to obtain the isovolumic difference in LVEDP (DeltaLVEDP). In all dogs, the technique yielded values of pericardial pressure equal to DeltaLVEDP as well as equal to that measured using a previously placed balloon transducer in the same location and at the same A(LV). We conclude that, when the pressure of the pericardial liquid is appropriately measured, it (in addition to the balloon-measured contact stress) defines the diastolic constraining effect of the pericardium. Furthermore, we suggest that earlier measurements of pericardial "liquid pressure" were low, due to an artifact of measurement.     

Strain-gauge plethysmographic measurement of the systemic bioavailability of oral nitroglycerin in liver cirrhosis

7 cirrhotic (M = 3, F = 4, mean age 55, range 35-74) and 7 healthy subjects (M = 6, F = 1, mean age 24, range 23-40) were studied. 2.5mg% nitroglycerin were administered per os. This drug is quite completely metabolized in its first pass through the liver (first pass effect). Peripheric vascular effect of nitroglycerin was evaluated by venous occlusion strain-gauge plethysmography, ECG-coupled (Rest Flow measurement RF, in ml/min/100 ml). No statistically significant differences were found between pre-drug RF in the two groups and between pre and post-drug measurements in healthy subjects. Post-drug RF decreased in cirrhotic subjects when compared either to pre-drug values or to post-drug values in normal subjects (statistically significant after the third minute, p ranging less than 0.05 and less than 0.001). The different peripheric vascular effect found in the two groups was considered as a consequence of the increased drug bioavailability in cirrhotics, caused by portosystemic shunts.     

A radiometric technique for the measurement of adenylosuccinate lyase

When radioactive adenylsuccinic acid (AMP-S) is metabolized to AMP and fumaric acid by the enzyme adenylsuccinate lyase (EC 4.3.2.2), a proton is released to the solvent as ³H₂O. This removal is believed to be stereospecifically identical to that catalyzed by the enzyme, l-aspartase [1–5], and therefore entails the loss of a proton from C-3 of the dicarboxylic acid moiety of the nucleotide. Advantage has been taken of this fact in the design of a facile assay for this enzyme. Adenylosuccinic acid, tritiated on C-2 and C-3 of the l-aspartase moiety, is prepared by chemical synthesis. This product is purified, lyophilized to dryness and reconstituted in a solution of unlabelled AMP-S, bringing the final concentration to 5·10^?3 M, and the final specific activity to 8.0 μCi/mol. 5-μl aliquots of this substrate are then incubated at 37°C with 5-μl aliquots of tissue extract; after an appropriate period, any tritium released to the solvent water is distilled at room temperature overnight into a 5 μl droplet of saturated aqueous KOH adherent to the lid of the sealed reaction vessel. The lid is removed and tritium thereon is measured by scintillation spectrometry. The assay, performed as prescribed, is facile, in that it permits the simultaneous estimation of the lyase activity in a large battery of samples, is not interfered with by opalescent or proteinaceous suspensions, is accurate and outstandingly sensitive.