Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.     

Real-time oligonucleotide hybridization kinetics monitored by resonant mirror technique

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.     

Customized k-nearest neighbourhood analysis in the management of adolescent idiopathic scoliosis using 3D markerless asymmetry analysis

Adolescent Idiopathic Scoliosis (AIS) is a 3D spinal deformity characterized by curvature and rotation of the spine. Markerless surface topography (ST) analysis has been proposed for diagnosing and monitoring AIS to reduce the X-ray radiation exposure to patients. This method captures scans of the cosmetic deformity of the torso using visible, radiation-free light. The asymmetry analysis of the torso, represented as a deviation contour map with deviation patches outlining the areas of cosmetic asymmetries, has previously been shown to predict the severity and progression of the condition in comparison with radiographs, by using classification trees. While the classification results were promising, it was reported that some mild curves were erroneously diagnosed. Furthermore, this approach is highly sensitive to threshold values selected in the decision trees. Therefore, this study aims to define a custom Neighbourhood Classifier algorithm for AIS classification to improve the accuracy, sensitivity, and specificity of predicting curve severity and curve progression in AIS. Curve severity was predicted with 80% accuracy (sensitivity = 81%; specificity = 79%) for thoracic-thoracolumbar curves and 72% (sensitivity = 93%; specificity = 53%) for lumbar curves. This represents an improvement over the previous method with curve severity accuracies of 77% and 63% for thoracic-thoracolumbar and lumbar curves, respectively. Additionally, curve progression was predicted with 93% accuracy (sensitivity = 83%; specificity = 95%) representing a substantial improvement over the previous method with an accuracy of 59%. The current method has shown the potential to further reduce radiation exposure for AIS patients by avoiding X-rays for mild and non-progressive curves identified using ST analysis.     

Melting curves on less than 1 μg of nucleic acid

Two types of microcuvettes were developed for the registration of melting curves in normal uv-spectrophotometers. Nucleic acid (0.5–1 μg) is required to determine changes in absorption with an accuracy of 0.1 mA. The quartz cuvette allows the registration of equilibrium melting curves up to 100°C. The other type of cuvette made from gilded brass allows to measure slow temperature jumps up to 80°C and equilibrium melting curves up to 70°C. The time range of ～3 s to several hours is accessible complementing the Eigen/De Mayer temperature-jump technique (1963, in Techniques of Organic Chemistry, Vol. VIII/2, pp. 895–1054, Wiley, New York). Temperature jumps of high amplitude as well as jumps to lower temperatures are possible. Examples of measurements on viroids and double-stranded viral RNA are presented.     

An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm

To measure cytochrome P-450 (P-450) content in hepatocyte cytoplasm, we developed a dual monochromator-equipped microphotometry system (KWSP-1). Simultaneous measurements of absorbance at 450 and 490 nm with narrow band width (0.5 nm) and small spot size (2 microns) were accomplished by this system. Corresponding fields in serial sections could be easily and rapidly identified under the Nomarski imaging mode of KWSP-1. Photometric accuracy and repeatability of wavelength setting of KWSP-1 were also satisfactory for measurement of P-450. With this system, it is thus possible to measure the extinction of P-450 from many small measuring areas and to precisely determine P-450 content in the cytoplasm of rat hepatocytes. A microphotometric method was developed using cuvette slides and two serial 10-microns thick sections (mapping method). The intracellular distribution of P-450 in individual hepatocytes could be visualized by the mapping method with KWSP-1. However, this method was not applicable to tissue sections containing hemoglobin larger than 4 microM.     

Changes in heart rate and circadian cardiac rhythm as physiological biomarkers for estimation of functional state of crayfish <Emphasis Type="Italic">Pontastacus leptodactylus</Emphasis> Esch. upon acidification of the environment

The changes in heart rate and circadian cardiac rhythm of crayfish Pontastacus leptodactylus Esch. kept in a lightning regime that is close to natural under optimal or low pH values were studied. The heart rate was registered in real time using an original noninvasive fiberoptic method. Upon acidification, disorders in circadian cardiac rhythm and organism reaction (by heart rate) in the suspension test were detected. The characteristics of cardiac activity are considered criteria for estimating the crayfish’s functional state at normal and stress conditions caused by the changes in the quality of the environment.     

基于通量塔净生态系统碳交换数据的植被物候遥感识别方法评价

选择北美洲72座通量塔观测的净生态系统碳交换（NEE）数据来计算植被物候,并以此作为参考数据,从可行性和准确性两方面对阈值法、移动平均法和函数拟合法三大类常用的植被物候遥感识别方法进行了综合评价.结果表明： 基于局部中值的阈值法对植被物候识别的可行性和准确性均最优;其次为Logistic函数拟合法中的一阶导数方法;移动平均法对植被物候识别的可行性和准确性与移动窗口的大小有关,对于16 d合成的归一化差值植被指数（NDVI）时间序列数据来说,移动窗口大小为15时能获得较优的结果;而全局阈值法对植被物候识别的可行性和准确性均最差;Logistic函数拟合法中的曲率变化率方法在识别植被物候时虽然与基于NEE数据得到的植被物候在数值上存在较大偏差,但二者之间具有较高的相关性,说明基于曲率变化率方法识别出的植被物候能较真实地反映植被物候在时空上的变化趋势.     

The "systolic volume balance" method for the noninvasive estimation of cardiac output based on pressure wave analysis

Cardiac output (CO) monitoring is essential for the optimal management of critically ill patients. Several mathematical methods have been proposed for CO estimation based on pressure waveform analysis. Most of them depend on invasive recording of blood pressure and require repeated calibrations, and they suffer from decreased accuracy under specific conditions. A new systolic volume balance (SVB) method, including a simpler empirical form (eSVB), was derived from basic physical principles that govern blood flow and, in particular, a volume balance approach for the conservation of mass ejected into and flowed out of the arterial system during systole. The formulas were validated by a one-dimensional model of the systemic arterial tree. Comparisons of CO estimates between the proposed and previous methods were performed in terms of agreement and accuracy using "real" CO values of the model as a reference. Five hundred and seven different hemodynamic cases were simulated by altering cardiac period, arterial compliance, and resistance. CO could be accurately estimated by the SVB method as follows: CO = C × PP(ao)/(T - P(sm) × T(s)/P(m)) and by the eSVB method as follows: CO = k × C × PP(ao)/T, where C is arterial compliance, PP(ao) is aortic pulse pressure, T is cardiac period, P(sm) is mean systolic pressure, T(s) is systolic duration, P(m) is mean pressure, and k is an empirical coefficient. SVB applied on aortic pressure waves did not require calibration or empirical correction for CO estimation. An empirical coefficient was necessary for brachial pressure wave analysis. The difference of SVB-derived CO from model CO (for brachial waves) was 0.042 ± 0.341 l/min, and the limits of agreement were -0.7 to 0.6 l/min, indicating high accuracy. The intraclass correlation coefficient and root mean square error between estimated and "real" CO were 0.861 and 0.041 l/min, respectively, indicating very good accuracy. eSVB also provided accurate estimation of CO. An in vivo validation study of the proposed methods remains to be conducted.     

A versatile microstirrer and oxygen electrode system for a spectrophotometer cuvette.

A microstirring device is described which measures only 1 × 1 × 0.4 cm outsidedimensions and is thus suitable for insertion beneath most spectrophotometer cuvettes without displacing the cuvette from the light path and without the need for modification of the spectrophotometer. The top of the cuvette remains accessible for a specially designed cuvette lid and electrode. This combined equipment is particularly suitable for the simultaneous measurement of two parameters, one with the electrode and the other by spectrophotometry. The design of the lid also permits the addition of reactants, including O₂, during monitoring. The equipment has been used to measure the oxygen-binding isotherm of human hemoglobin using 0.1 mg of protein. The curve so obtained was identical with curves found in the literature and obtained under the same conditions of pH, temperature, etc.     

A self-calibrating telemetry system for measurement of ventricular pressure-volume relations in conscious, freely moving rats

Using Bluetooth wireless technology, we developed an implantable telemetry system for measurement of the left ventricular pressure-volume relation in conscious, freely moving rats. The telemetry system consisted of a pressure-conductance catheter (1.8-Fr) connected to a small (14-g) fully implantable signal transmitter. To make the system fully telemetric, calibrations such as blood resistivity and parallel conductance were also conducted telemetrically. To estimate blood resistivity, we used four electrodes arranged 0.2 mm apart on the pressure-conductance catheter. To estimate parallel conductance, we used a dual-frequency method. We examined the accuracy of calibrations, stroke volume (SV) measurements, and the reproducibility of the telemetry. The blood resistivity estimated telemetrically agreed with that measured using an ex vivo cuvette method (y=1.09x - 11.9, r2= 0.88, n=10). Parallel conductance estimated by the dual-frequency (2 and 20 kHz) method correlated well with that measured by a conventional saline injection method (y=1.59x - 1.77, r2= 0.87, n=13). The telemetric SV closely correlated with the flowmetric SV during inferior vena cava occlusions (y=0.96x + 7.5, r2=0.96, n=4). In six conscious rats, differences between the repeated telemetries on different days (3 days apart on average) were reasonably small: 13% for end-diastolic volume, 20% for end-systolic volume, 28% for end-diastolic pressure, and 6% for end-systolic pressure. We conclude that the developed telemetry system enables us to estimate the pressure-volume relation with reasonable accuracy and reproducibility in conscious, untethered rats.     

Prediction of VO2max during cycle exercise in pregnant women

We measured maximal O2 uptake (VO2max) during stationary cycling in 40 pregnant women [aged 29.2 +/- 3.9 (SD) yr, gestational age 25.9 +/- 3.3 wk]. Data from 30 of these women were used to develop an equation to predict the percent VO2max from submaximal heart rates. This equation and the submaximal VO2 were used to predict VO2max in the remaining 10 women. The accuracy of VO2max values estimated by this procedure was compared with values predicted by two popular methods: the Astrand nomogram and the VO2 vs. heart rate (VO2-HR) curve. VO2max values estimated by the derived equation method in the 10 validation subjects were only 3.7 +/- 12.2% higher than actual values (P greater than 0.05). The Astrand method overestimated VO2max by 9.0 +/- 19.4% (P greater than 0.05), whereas the VO2-HR curve method underestimated VO2max by only 1.6 +/- 10.3% in the same 10 subjects (P greater than 0.05). Both the Astrand and the VO2-HR curve methods correlated well with the actual values when all 40 subjects were considered (r = 0.77 and 0.85, respectively), but the VO2-HR curve method had a lower SE of prediction than the Astrand method (8.7 vs. 10.4%). In a comparison group of 10 nonpregnant sedentary women (29.9 +/- 4.5 yr), an equation relating %VO2max to HR nearly identical to that obtained in the pregnant women was found, suggesting that pregnancy does not alter this relationship. We conclude that extrapolating the VO2-HR curve to an estimated maximal HR is the most accurate method of predicting VO2max in pregnant women.     

Construction and Comparison of Two Receiver Operating Characteristic Curves Derived from the Same Samples

Receiver operating characteristic (ROC) curves are used to describe the performance of diagnostic procedures. This paper proposes a simple method for the statistical comparison of two ROC curves derived from the same set of patients and the same set of healthy subjects. Generalization to studies involving more than two screening factors is straightforward. This method does not require the calculation of variances of the areas or difference of areas under the curves.     

Toward optical spectroscopy-guided lung biopsy: Demonstration of tissue-type classification

The diagnostic yield of standard tissue-sampling modalities of suspected lung cancers, whether by bronchoscopy or interventional radiology, can be nonoptimal, varying with the size and location of lesions. What is needed is an insitu sensor, integrated in the biopsy tool, to objectively distinguish among tissue types in real time, not to replace biopsy with an optical diagnostic, but to verify that the sampling tool is properly located within the target lesion. We investigated the feasibility of elastic scattering spectroscopy (ESS), coupled with machine learning, to distinguish lung lesions from the various nearby tissue types, in a study with freshly-excised lung tissues from surgical resections. Optical spectra were recorded with an ESS fiberoptic probe in different areas of the resected pulmonary tissues, including benign-margin tissue sites as well as the periphery and core of the lesion. An artificial-intelligence model was used to analyze, retrospectively, 2032 measurements from excised tissues of 35 patients. With high accuracy, ESS was able to distinguish alveolar tissue from bronchi, alveolar tissue from lesions, and bronchi from lesions. This ex vivo study indicates promise for ESS fiberoptic probes to be integrated with surgical intervention tools, to improve reliability of pulmonary lesion targeting.     

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin- resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to - 1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prediction of maximum expiratory flow rate from area-transmural pressure curve of compressed airway.

The site of greatest airway deformation in dog lungs was located during maximum expiratory flow by use of tantalum bronchography, fiberoptic bronchoscopy, and airway pressure measurements. A series of area vs. transmural pressure curves for each of these segments of the airway was produced after stepwise changes in transmural pressure. Measurements of area were made using cinephotography to elucidate the effect of time on airway compliance. The maximum flow rate was calculated using the t = 0.1 s compliance curve of the airway. An equation was derived so that maximum flow (V) could be calculated from the area (A) and transmural pressure (Ptm) of the flow-limiting segment. This equation, V = K-A square root of Ptm, implied that if V were constant then A must vary as Ptm-1/2. It was demonstrated that the area-transmural pressure curve of the flow-limiting segment showed this relationship between A and Ptm and that the flow calculated from this equation and the data from the A-Ptm curve gave flows identical to those measured during maximum expiration. The phenomena of effort-independent flow and negative effort dependence are also explained in terms of the area-transmural pressure curve of the flow-limiting segment.     

Maximum shortening velocity of smooth muscle: zero load-clamp vs. afterloaded method

Previous reports from this laboratory of force-velocity relationships of canine tracheal smooth muscle (TSM) have presented maximum shortening velocities (Vmax) mathematically derived from the linearized transformation of the Hill equation (A. V. Hill, Proc. Roy. Soc. London, Ser. B., 126:136-195, 1938). Recent technical advances enable us to measure Vmax directly using an electromagnetic lever system that can instantaneously clamp to a zero load, thus we compared values of Vmax derived mathematically and those directly measured on the same TSM strips. Derived Vmax values from afterloaded isotonic shortening curves for loads greater than preload were 0.328 +/- 0.021 optimal length (lO)/s and were not significantly different from zero load-clamp measurements of 0.301 +/- 0.022 lO/s from the same (n = 15) muscles. These data indicate that Vmax values mathematically derived for TSM from conventional isotonic afterloaded force-velocity curves are valid estimates of zero load velocity, because they were not significantly different from values obtained by direct measurement using the zero load-clamp technique.     

Estimation of cardiac output from dynamic contrast multispiral tomographic data

OBJECTIVE: To describe and apply a method for programmatically calculating central hemodynamic parameters from the data of dynamic contrast X-ray computed tomography (CT). MATERIAL AND METHODS: A procedure is proposed to calculate cardiac output (CO) on the basis of the tracer dilution method as a ratio of the injected dose of a contrast agent (Urografin, Shering AG, Austria) to the area under the concentration-time curve derived for one of the cardiac chambers: [see text]. The blood concentration of the agent was estimated from the blood iodine (I) concentration-X-ray density (XD) regression dependence: [XD] = 0.49+22.07 x [I]. Thirty-three patients were studied on a multispiral X-ray Somatom Sensation 4 computed tomograph (Siemens), by injecting 15-20 ml of Urografin. The calculating method was realized as a software package for processing dynamic contrast CT data. RESULTS: The proposed method could exactly determine the values of cardiac output: the correlation with the results of the reference technique (Doppler echo measurement) was highly significant (r = 0.95; p < 0.001) in the whole range of values. Moreover, the data on XD could accurately determine the blood concentration of both iodine (I): [XD] = 0.49+22.07 x [I] and gadolinium as a component of the contrast paramagnetic Gd-DTPA for which the solution concentration-XR dependence was as follows: XD 1.2+33.6 x [Gd]. CONCLUSIONS: Thus, dynamic spiral CT may be also used for the estimation of cardiac output by the tracer dilution method.     

Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules

A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation.     

Cholesterol turnover and metabolism in two patients with abetalipoproteinemia

Total body turnover of cholesterol was studied in two patients with abetalipoproteinemia, a 32-year-old man and a 31-year-old woman. The patients received [14C]cholesterol intravenously, and the resulting specific activity-time curves (for 40 and 30 weeks, respectively) were fitted with a three-pool model. Parameters were compared with those from studies of cholesterol turnover in 82 normal and hyperlipidemic subjects. A three-pool model gave the best fit for the abetalipoproteinemic patients, as well as for the 82 previously studied subjects, suggesting general applicability of this model. Cholesterol production rates in the two abetalipoproteinemic subjects (0.82 and 0.89 g/day) were close to values predicted for persons of their body weight. Thus, total body turnover rate of cholesterol was quite normal in abetalipoproteinemia, confirming previous reports. Very low values (9.2 and 8.4 g) were found for M1, the size of the rapidly exchanging compartment pool 1, in the two abetalipoproteinemic subjects. These values were well below the values predicted (from the comparison study population) for normal persons of this size with low plasma cholesterol levels. For one patient, total body exchangeable cholesterol was very low, although not significantly below the predicted values for a person of his size. In the second patient, the observed estimate for total body exchangeable cholesterol was well within the range of values predicted for persons of her size with low to extremely low cholesterol levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

In situ measurements of alternative respiratory pathway activity are needed to provide insight into the energy efficiency of plant metabolism under various conditions in the field. The only reliable method at present to measure alternative oxidase (AOX) activity is through measurement of changes in delta(18)O(O(2)), which to date has only been used in laboratory environments. We have developed a cuvette system to measure partitioning of electrons to AOX that is suitable for off-line use and for field experiments. Plant samples are enclosed in airtight cuvettes and O(2) consumption is monitored. Gas samples from the cuvette are stored in evacuated gas containers until measurement of delta(18)O(O(2)). We have validated this method using differing plant material to assess AOX activity. Fractionation factors were calculated from delta(18)O(O(2)) measurements, which could be measured with an accuracy and precision to 0.1 per thousand and 0.3 per thousand, respectively. Potential sources of error are discussed and quantified. Our method provides results similar to those obtained with laboratory incubations on-line to a mass spectrometer but greatly increases the potential for adoption of the stable isotope method.