Instrumentation simultaneously measuring VCO2 and VO2 in humans using titration methods

An instrument has been developed for the simultaneous measurement of carbon dioxide excretion (VCO2) and oxygen uptake (VO2). This instrument, the Nutrimeter, gives these breath-averaged measurements continuously without having to determine respiratory flow rate, perform timed spirometric gas collections, or determine absolute CO2 or O2 concentrations. It can be used on ventilated or nonventilated patients in long- and short-term studies. VO2 is determined via the replenishment technique. VCO2 is determined via a new technique, absorption-titration, described here. Bench test results of VCO2 measurements show a standard error of the estimate (SEE) +/- 0.591% of full scale (500 ml/min) and maximum single point error (MSPE) of +/- 3.54% over a 100--350 ml/min range. VO2 measurements show SEE +/- 0.518% of full scale (1,000 ml/min) and MSPE +/- 2.42% over a 100--450 ml/min range. In 31 human clinical trials the Nutrimeter was compared with the open-circuit spirometric collection and micro-Scholander analysis technique. VCO2 measurements show SEE +/- 2.208% and MSPE +/- 10.57% over 135--315 ml/min. VO2 measurements show SEE +/- 1.134% of full scale and MSPE +/- 9.54% over 170--360 ml/min. Response time is 60 s optimally for step changes in VO2 (0--90% of steady-state value), 90 s for VCO2.     

On-line titration method for monitoring buffer capacity and total volatile fatty acid levels in anaerobic digesters

The construction and use of an automatic on-line titration unit for routine or event- initiated monitoring of alkalinity, buffer capacity, and volatile fatty acid (VFA) levels is presented. Under computer control a sample of digester liquor is pumped into the titration vessel and weighed. A sequence of titration, sparging, and back-titration operations are then initiated during which the pH and weight are recorded continuously and a titration curve constructed. From the curve, estimates of the alkalinity, buffer capacity to any desired pH endpoint, and total VFA levels are computed. The data is stored to disk and output as hard copy together with the titration curve itself. Monitoring and control of the titration apparatus is effected by a microcomputer via two analog input lines and eight digital output lines, respectively. The system is suitable for downloading to a small, inexpensive dedicated microprocessor-based system. The apparatus is constructed from standard and widely available equipment and the titration sequence, being under software control, is fully adaptable to particular requirements. The use of this facility in the on-line monitoring, control and optimization of the anaerobic digestion process is discussed.     

Autointerference in silver accumulation in macrophages without affecting phagocytic, migratory or interferon-producing capacity

Silver accumulation and processing in mouse peritoneal macrophages was studied in vitro by autometallographic visualization of intracellular silver. During the first 24 h of incubation in a medium containing from 5 microM to 20 microM of silver lactate, an inverse relationship between silver concentration in the former and visualizable silver in macrophages was recorded. Later, however, the cells treated with higher silver concentrations accumulated most silver. Cells exposed to silver concentrations above these levels exhibited acute coagulation necrosis and disintegrated within the first 15 min of silver treatment. Macrophages treated with silver lactate concentrations not causing acute cytotoxicity showed no impairment of their phagocytic, migratory or interferon-producing capacities. The significance of autointerference in silver accumulation and processing in macrophages is discussed, and a functional defect in the lysosome/phagosome system is suggested as a basis for the phenomenon.     

An active-site titration method for lipases

A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.     

A practical and reliable method for measuring ethane and pentane in expired air from humans.

We describe a method for the collection of expired air and further document the performance of our analytical technique that is used to measure ethane and pentane simultaneously. Four minutes of breathing hydrocarbon-free air before collection effectively removed high concentrations of residual ambient ethane and pentane from the lungs, with washout times up to 30 min resulting in no further reductions in breath hydrocarbons. Mean (+/-SE) exhalation rates (pmol/kg b.wt./min) in 11 subjects were 2.4 +/- 0.6 for ethane and 1.5 +/- 1.3 for pentane. Total intraindividual variability in exhalation rates (as percent coefficient of variation, %CV), measured from 4 subjects on at least 6 different days, was greater for pentane (44% CV) than for ethane (29% CV). Analytical variability contributed 6% to the total %CV. Advantages of the method are described, and reasons for the large variability in values reported in the literature are discussed.     

A method for measuring bacterial chemotaxis parameters in a microcapillary

A new method was developed which enables chemotaxis parameters to be measured at a single-cell level inside a capillary for the first time. The chemotaxis chamber consists of two reservoirs communicating through a capillary tube 50 mum in diameter. Chemotaxis parameters are measured inside the capillary using image analysis, after a nearly linear attractant concentration gradient has been generated along the capillary by diffusion. Compared to previously published techniques, this method provides a well-characterized chemoattractant concentration profile in addition to allowing single-cell parameters to be measured inside a fine capillary. This procedure was used to measure the single-cell chemotaxis parameters for Escherichia coli K12, and the results are compared to published data on single E. coli cells chemotaxing in bulk. (c) 1996 John Wiley & Sons, Inc.     

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.     

Histone-like protein: a novel method for measuring stress in fish

We assessed the effect of chronic stress using a group of potent, broad-spectrum antimicrobial polypeptides, called histone-like proteins (HLPs), which appear to be an important component of non-specific immunity in channel catfish Ictalurus punctatus skin. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the predominant HLP (HLP-1) in channel catfish skin. Catfish were then exposed to a chronic stress consisting of overcrowding and elevated ammonia. Healthy unstressed fish had consistently high HLP-1 levels, but fish that had been stressed for 1 wk had significantly depressed HLP-1 levels; HLP-1 levels declined further in fish stressed for 3 or 4 wk. The time-dependent decline in HLP-1 levels was not accompanied by any gross signs of disease. In contrast to HLP-1 levels, antibacterial activity in the skin was significantly greater in fish stressed for 1 wk compared with unstressed fish; in addition, antibacterial activity was the same in fish that were unstressed or stressed for 3 or 4 wk. This suggests that other antibiotics besides HLP-1 may be induced in the skin, especially during early stages of stress, that may compensate for depressed HLP-1 levels. Our results indicate that chronic stress has a significant suppressive effect on HLP-1 levels in channel catfish skin. The reduction of HLP-1 in the absence of clinical signs of disease, combined with evidence that its levels are not affected by the acute stressors of capture or sampling, suggests that HLP levels may be a promising indicator for monitoring fish health.     

A steady state method of measuring carbon monoxide diffusing capacity of the lung of sheep

A method for measuring the diffusing capacity of the lung for carbon monoxide (DLCO) in sheep was developed. The test's usefulness and reliability was studied in ten, splenectomized adult ewes. Hemoglobin concentration and weight were found to affect sheep DLCO. This article describes a method of determining DLCO in sheep, gives preliminary results of limited testing, and discusses factors affecting DLCO in sheep.     

Increased working capacity with hyperoxia in humans

The purpose of the study was to examine the influence of oxygen-breathing on maximal oxygen uptake (VO2max) and submaximal endurance performance. Six young women and five men rode a cycle-ergometer while breathing compressed air (normoxia, NOX) or a 55% O2 in N2 mixture (hyperoxia, HOX). The VO2max increased significantly by 12% (P less than 0.01) with HOX in the women but not in the men (+4%; nonsignificant). Maximal heart rate was also increased with HOX in the women but not in the men. Endurance time during work to exhaustion at 80% of normoxic VO2max was 41% longer in HOX than in NOX (P less than 0.025) with no significant difference between the men and the women. The variation among individuals was large. The oxygen uptake and respiratory quotient were not different in the two endurance tests, but pulmonary ventilation (VE) and blood lactate concentration were lower in HOX than in NOX, especially during the latter part of the task. Plasma base deficit (BDpl) increased initially by 3.5 mmol.l-1 during HOX and then stabilized. In NOX, a continuous increase was seen and the change was more than twice as large. Relative to BDpl, VE was higher in HOX than in NOX indicating a more efficient ventilatory compensation of the metabolic acidosis. The reduced ventilatory demand and lower metabolic acidosis in HOX in combination with lower relative exercise intensity may have contributed to the longer time to exhaustion. However, the pattern of individual variation suggested that other mechanisms were also involved.     

Beer increases plasma antioxidant capacity in humans

The positive association of a moderate intake of alcoholic beverages with a low risk for cardiovascular disease, in addition to ethanol itself, may be linked to their polyphenol content. This article describes the effect of acute ingestion of beer, dealcoholized beer, and ethanol (4.5% v/v) on the total plasma antioxidant status of subjects, and the change in the high performance liquid chromatography profile of some selected phenolic acids (caffeic, sinapic, syringic, and vanillic acids) in 14 healthy humans. Plasma was collected at various times: before (T0), 1 hour after (T1), and 2 hours after (T2) drinking. The study is part of a larger research planned to identify both the impact of brewing on minor components potentially present in beer and their metabolic fate in humans. Beer was able to induce a significant (P < 0.05) increase in plasma antioxidant capacity at T1 (mean +/- SD: T0 1,353 +/- 320 microM; T1 1,578 +/- 282 microM), returning close to basal values at T2. All phenolic acids measured in plasma tended to increase after beer intake (20% at T1, 40% at T2). Syringic and sinapic acid reached statistical significance (P < 0.05 by one-way analysis of variance-Fisher's test) at T1 and T2, respectively. Plasma metabolic parameters (glucose, total cholesterol, triglycerides, and uric acid) and plasma antioxidants (alpha-tocopherol and glutathione) remained unchanged. Ethanol removal impaired the absorption of phenolic acids, which did not change over the time of the experiment, accounting for the low (and not statistically significant) increase in plasma antioxidant capacity after dealcoholized beer drinking. Ethanol alone did not affect plasma antioxidant capacity or any of the antioxidant and metabolic parameters measured.     

Protocol for measuring the endurance capacity of mice in an adjustable-current swimming pool

We re-examined the methods used in an adjustable-current swimming pool for evaluating the endurance capacity of mice to improve the sensitivity and reproducibility. We found that the BALB/c strain was most suitable to minimize the variation in time taken to swim to the point of fatigue. We found that precise adjustment of the apparatus and the use of three primary swimming trials to select mice before a study made the swimming time more uniform. This procedure was used to establish a more precise evaluation protocol.