Washout Procedure Research Articles

Quantification of the total Na,K-ATPase concentration in atria and ventricles from mammalian species by measuring3H-ouabain binding to intact myocardial samples. Stability to short term ischemia reperfusion

Na,K-ATPase concentration was measured by vanadate facilitated 3H-ouabain binding to intact samples taken from various parts of porcine and canine myocardium. In porcine and canine heart 3H-ouabain binding site concentration in ventricles was 1.4-2.5 times larger than in atria. Evaluation of 3H-ouabain binding kinetics revealed no major difference between atria and ventricles: Equilibrium was obtained after the same incubation time in right atrium (RA) as in left ventricle (LV), both in porcine and canine heart. Unspecific uptake and retention of 3H-ouabain was for porcine heart RA and LV 1.5 and 1.4, respectively, and for canine heart RA and LV, both 1.2% filling (i.e., volume (ml) of incubation medium 3H-radioactivity taken up per mass unit (g wet wt.) of tissue multiplied by 100). The apparent dissociation constant (KD) was 1.4 x 10(-8) and 1.9 x 10(-8) in porcine RA and LV and 2.6 x 10(-8) and 6.1 x 10(-8) mol/l in canine RA and LV. Loss of specifically bound 3H-ouabain during the washout procedure occurred with a half-life time (T1/2) of 16.7 and 28.6 in RA and LV of porcine heart and 91.2 and 151.6 h in RA and LV of canine heart. Duly corrected for these errors of the method--factor 1.16 and 1.13, respectively, for porcine RA and LV, and factor 1.11 and 1.13 for canine RA and LV, total 3H-ouabain binding site concentration was found to be 553 +/- 74 and 1037 +/- 45 pmol/g wet wt. (means +/- SEM, n = 5) in porcine RA and LV, and 569 +/- 37 and 1410 +/- 40 pmol/g wet wt. (means +/- SEM, n = 5) in the canine RA and LV. These values were confirmed by measurements of 3H-digoxin binding to the porcine heart. The present quantification of myocardial Na,K-ATPase gives values up to 154 times higher than measurements based upon Na,K-ATPase activities in membrane fractions where the recovery of Na,K-ATPase may be less than 1% due to loss during purification. A higher Na,K-ATPase concentration is found in small animals than in large animals. A relationship between higher concentration of Na,K-ATPase and larger pressure work in ventricles compared to atria is suggested. Myocardial 3H-ouabain binding sites were found to be stable for 20 min of ischemia, followed by 1 h of reperfusion, supporting the concept that myocyte injury induced by short term ischemia may be reversible and that reperfusion may result in normalization.

Tracheal pressure and impedance as determinants of gas exchange during high frequency ventilation

This work explores the use of catherer pressure transducers to measure time averaged, mean squared pressure (MSP) in the trachea of anesthetized animals during high frequency ventilation using a prototype Emerson oscillator with a 25 ml stroke volume at 15 and 34 Hz. Tidal volume and MSP were varied by shunting a portion of the ventilator output for the study of the effect of driving pressure on ventilation. A given level of ventilation was accomplished at a lowere driving pressure at the lowere frequency. Both ventilation as assessed by a modified nitrogen washout procedure and gas exchange were related to MSP and oscillatory frequency by the substitution of MSP/Z 2 for alveolar ventilation in the equations of Jaeger et al. (1984). The technique and equations describing it were validated by the prediction of impedance changes resulting from shifts in oscillatory frequency. The calculated impedances matched experimentally measured values found in dogs by other investigators. Catherter pressure transducers are thus shown to be a useful tool for quantifying high frequency ventilation when coupled with a knowledge of respiratory impedance. The technique offers advantages over existing techniques, the most being notable ease of use due to the catheters' exceptional frequency response.

Effects of chlorpromazine and its dihydroxy metabolite on calcium movement and electrical activity in superior cervical ganglia.

Chlorpromazine (CPZ) binding and the binding to isolated guinea pig superior cervical of a toxic metabolite 7,8-dihydroxychlorpromazine (7,8-diOH-CPZ) were studied under resting and potassium-stimulated conditions. Maximal binding occurred more rapidly in high potassium, with binding of CPZ 10 fold higher than that of 7,8-diOH-CPZ. However, in low potassium media 50% of bound CPZ and 60% of 7,8-diOH-CPZ was retained following a 240-min washout procedure. When the washout of the drugs from the tissue was studied, release was found to be more extensive in high potassium washout media. Under resting conditions, at 10 microM CPZ or 7,8-diOH-CPZ, calcium accumulation by ganglia decreased but increased at higher drug concentrations. Calcium (Ca) uptake increased with both drugs in high potassium medium. Ca efflux from the tissue was accelerated by both phenothiazines in a dose-dependent fashion. CPZ blocked ganglionic transmission at 20 microM while 7,8-diOH-CPZ (up to 100 microM) increased ganglionic potential amplitude. Thus CPZ and 7,8-diOH-CPZ differ in binding to ganglia and in effects on ganglionic transmission.

Respiratory mechanics in adolescents with idiopathic scoliosis.

Mechanisms causing the reduction in lung capacity commonly found in adolescents with idiopathic scoliosis (IS) have not been understood. In 29 patients with typical thoracic curvatures of mild to moderate degree (less than 60 degrees), total lung capacity (TLC) was a mean 75 +/- 13% (SD) of predicted. The patients could generate only -70 +/- 26 cm H2O (SD) maximal inspiratory airway pressure at function residual capacity, as compared with -102 +/- 28 cm H2O in 21 normal control subjects (p less than 0.001). Studies of lung mechanics in 15 of the patients showed that maximal transpulmonary pressure at TLC was also reduced. Static pressure volume curves were shifted to the right, and both static and dynamic lung compliance were significantly reduced. Although both upstream conductance per TLC and anatomic dead space per TLC were abnormally high, relationships between maximal expiratory flow and static lung recoil were appropriate for age, indicating a normal growth of airway dimensions. From results of the single-breath nitrogen washout procedure, amounts of trapped nitrogen were also normal, indicating that the low lung compliance is not caused by airway closure. After a 5-min period of positive pressure (25 cm H2O) breathing, dynamic compliance increased by a mean of 34% in subjects with low TLC, and by a significantly smaller (p less than 0.05) mean increase of 14% in subjects with normal TLC. The 15 patients were restudied 1 yr after corrective surgery by the Harrington procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of Antibody-coated Bacteria and Bladder Washout Tests to Localize Infection in Spinal Cord Injury Patients

AbstractThe validity of the antibody-coated bacteria test and Fairley washout procedure to determine whether urinary tract infections were of kidney or bladder origin in spinal cord injury patients was assessed by comparing test results with those obtained from culture of ureteral urine specimens. It was concluded that neither the presence of antibody-coated bacteria nor a positive bladder washout test can be considered as definitive evidence that bacteriuria is of renal origin in these patients.

Neutral red stains ganglia in the vagal motor pathway to ferret trachea without affecting ganglionic transmission

To determine the effect of Neutral red (0.01%) on neural transmission through ganglia, we used an in vitro nerve-muscle preparation of ferret trachea. Before, during, and after incubating the trachea in Neutral red, we induced isometric muscle contractions first by activating preganglionic fibers with electrical stimulation of the vagus nerve, and then by activating postganglionic nerve fibers with electrical field stimulation. Incubation in Neutral red (0.01%) for 45 min at 38°C or 20°C reduced the responses to both pre- and postganglionic activation. When the control responses to pre- and postganglionic activation were matched, Neutral red depressed the 2 responses to the same degree, implying that the depression was confined to postganglionic structures. Washout of Neutral red from the medium restored the responses to both pre- and postganglionic activation. Histologic examination of all tissues proved that the ganglia were still stained after the washout procedure. We conclude that Neutral red (0.01%) depresses smooth muscle contractions evoked through neural pathways, and that this depression is reversible and confined to postganglionic structures, leaving ganglionic transmission intact.

Functional classification of chronic airflow limitation based on flow-volume and single-breath nitrogen washout criteria.

A simple means of functional classification of the individual patient has been devised. The performance of a forced flow-volume manoeuvre in concert with a single-breath nitrogen washout procedure in 54 comprehensively investigated patients with chronic airflow limitation yielded two parameters which could be employed in classification. A normalized alveolar nitrogen slope (FEV1/FVC X delta N2%) greater than 4.0% per litre clearly delineated emphysema. The peak flow ratio (PEFR/PIFR%) categorized other subgroupings: a value in excess of 100% strongly suggests primary bronchial disease, while a lower value almost certainly indicates a mixed functional disorder.

Helium and sulfur hexafluoride washout in asymmetric lung models.

Model calculations were carried out on extensible lung models that are characterized by asymmetries in branching pattern or cross section within the acinus. The calculations contribute to a better understanding of the intrapulmonary mixing of gases poorly soluble in blood. For single-breath washout stimulations of helium (He) and sulfur hexafluoride (SF6), only small differences were found between the results obtained from a lung model with an asymmetric branching pattern using either a local homogeneous or inhomogeneous delta V/V distribution. This indicates that the geometry of this model is the most important factor for generating realistic values for the slope of the alveolar plateau, the Bohr dead space and the end-tidal concentration ratio for SF6 and He. The calculated relation between the decrease in the slope of the alveolar plateau and postinspiratory breath-holding times showed good agreement with experimental findings in healthy adult subjects. Multiple-breath washout simulations enabled us to explain the experimentally observed high dilution of poorly soluble tracer gases in the end-tidal gas for the first few breaths during the washout procedure. Simulations of simultaneous multiple-breath washout experiments of He and SF6 showed that a shift in the crossover point of the normalized end-tidal washout curves is related in particular to cross-section asymmetry of the lung model. These asymmetric lung models are the only ones described up to now that explain many of the features of single- and multiple-breath washout experiments for gases poorly soluble in blood.

Flow distribution and cryoprotectant concentration in rabbit kidneys perfused with glycerol solutions

Rabbit kidneys were perfused at 10 °C with a solution containing gelatin polypeptides (Haemaccel), and glycerol was introduced, and then removed, using a technique that has previously been shown to result in viable kidneys. This involved increasing the concentration of glycerol in the perfusate from zero to a maximum of 3 m, holding it at this level for 30 min, and then decreasing it at the same rate to < 0.1 m. Measurements were made of the concentration of glycerol in cortex, cortico-medullary zone, and medulla at various stages of perfusion. During the experiments it was observed that vascular resistance increased dramatically toward the end of deglycerolization, and changes in regional perfusate flow were measured by the diffusable indicator method. It was found that renal tissue is effectively permeated by glycerol using this technique. The perfusate flow throughout all regions of the kidney was reduced during deglycerolization but the greatest effect was on cortico-medullary flow, which was found to be abnormally high during the initial stages of hypothermic perfusion, but was severely impaired when the glycerol was removed. The cryoprotectant was almost completely removed by the washout procedure adopted.

The effect of urinary glucose excretion on the plasma glucose clearances and plasma insulin responses to intravenous glucose loads in unanaesthesized dogs.

The effect of urinary glucose excretion on the plasma glucose clearance and insulin response to varying sizes of glucose loads was studied in normal unanaesthesized dogs. Glucose loads ranging from 0.15 to 1.25 g/kg b.w. were infused intravenously in a standard time period of 30 seconds. Plasma glucose and plasma immunoreactive insulin (IRI) concentrations were determined during one-hour after infusion. All urine excreted during the one-hour was collected by a catheterization and bladder wash-out procedure. The urinary glucose excretion was expressed as the percent of the glucose load. The urinary glucose excretion varied directly with the size of the glucose load and ranged from minimal to 12%. This would indicate that urinary losses play a considerable role proportionate to the degree of hyperglycaemia above the renal threshold. Thus, urinary loss of glucose must be recognized as an important factor infuencing the plasma glucose clearance and hence the plasma IRI response. A glucose load of 0.5 g/kg b.w. given in 30 seconds with the k-value calculated between 15-45 min would minimize the influence of urinary loss and provide more accurate plasma clearance values.

The effect of external calcium and lanthanum on platelet calcium content and on the release reaction

Calcium compartments in calf platelets were studied using a lanthanum washout procedure to distinguish between surface-bound calcium and intracellular calcium. The calcium content of calf platelets ranges from 20 to 60 nmol/10 9 platelets and is sensitive to the calcium concentration of the suspending medium. With 1 mM calcium in the medium, calcium uptake is rapid and reaches steady state within 1–2 min. Results obtained with the lanthanum procedure indicate that it is the surface compartment which is most affected by the extracellular calcium concentration. The surface compartment appears to be saturable and is highly exchangeable. Although the total calcium as well as the calcium content of the surface and internal compartments are variable, the ratio of calcium in either compartment to the total saturated calcium is quite constant. The data indicate that 68–85% of the platelet calcium is located internally. Thrombin produces an immediate release of platelet calcium and labeled serotonin and an increase in the 45Ca 2+ uptake of both the surface and internal compartments. The release reaction is not dependent upon exogenous calcium or an influx of exogenous calcium since it occurs even in the presence of ethyleneglycol-bis-(β-aminoethylether)-N,N′- tetraacetic acid. Lanthanum, however, inhibits the release reaction possibly by blocking surface calcium site and reducing the mobility of endogenous platelet calcium.

Distribution of ventilation during diaphragmatic breathing in obstructive lung disease.

The present study was designed to assess the effectiveness of voluntary diaphragmatic breathing on distribution of ventilation as measured by a nitrogen wash-out procedure and gamma camera imaging of the lung with radioactive xenon. In 11 patients with chronic obstructive lung disease, diaphragmatic breathing did not alter the indices of distribution calculated from analysis of the nitrogen wash-out curve. In 5 of 8 normal subjects, gamma camera imaging with radioactive xenon showed that initiation of inspiration from residual volume position by a diaphragmatic breathing maneuver directed the initial inspirate toward the lower zones in contrast to thoracic breathing, which directed it to the upper zones. In none of 5 patients with chronic obstructive lung disease was there a difference in topographic distribution of the initial inspirate from residual volume between the diaphragmatic and thoracic breathing maneuvers. It is suggested that in patients with chronic obstructive lung disease, good movement of ...

The correlation between the decline of the positive inotropic effect and the loss of cardiac glycosides from isolated atria during wash-out

The rate at which the positive inotropic effect of ouabain, digoxin and digitoxin diasppears from guinea-pig isolated atria during wash-out has been determined quantitatively. This rate was compared with the decrease in total tissue concentration during the wash-out procedure, previously established by means of tritium-labelled cardiac glycosides. The positive inotropic effect had entirely disappeared at 20–30 min; no important differences were observed between the glycosides studied. The decrease in total tissue concentration occurred more slowly. Calculations have shown that it is probably the rate at which the glycosides diffuse out of the extracellular space which governs the decrease in pharmacological effect. This disapearance of the positive inotropic action is not determined by the decline in total tissue concentration. The results suggest that the different duration of action of cardiac glycosides in vivo is caused by differences in elimination and/or metabolism and not by differences in fixation to heart muscle.

Ionic interaction with bone mineral I. Evidence for an isoionic calcium exchange with hydroxyapatite

Exchange of calcium with hydroxyapatite crystals in aqueous suspension was measured with 45Ca. The results were analyzed by fitting to a curve characterized by four exponential terms, of the form: Isotope uptake (or release) = σ i=I i=4A i e−α i t Total exchange was also calculated from measurements of non-isotopic calcium. Isotopic steady state was reached by 65 h. Uptake and release were characterized by similar A and α values. Release in 1 h was the same whether uptake had been carried out for 1 or 65 h. This, with the high values of α 1 and α 2, indicates that early exchange is quantitatively accounted for largely by the contribution of two ‘fast’ compartments. As further evidence for exchange, the uptake of isotope by hydroxyapatite was completely reversible by a wash-out procedure. Release was largely prevented by omission of Ca 2+ from solution, whether or not they were replaced with Na +; this indicates that calcium uptake is an isoionic exchange process.