Correlation of in vitro and in vivo models for the oral absorption of peptide drugs

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated. Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature. It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2) = 0.86; y = -196.22x + 1354.24), with rat oral bioavailability (R (2) = 0.64; y = -401.90x + 1268.86) as well as with human oral bioavailability (R (2) = 0.91; y = -359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2) = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs. Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.     

Dependence of transcutaneous O2 partial pressure on cutaneous blood flow

Transcutaneous PO2 was measured using a transcutaneous PO2 electrode heated to 45 degrees C on the forearm of 19 healthy volunteers. Cutaneous blood flow (CBF) was estimated indirectly from the heating power of the electrode (HP) and with an 8-MHz bidirectional ultrasonic probe by Doppler shift in a fingertip at 45 degrees C (DF). Blood flow was regulated by an upper arm cuff. Mean transcutaneous PO2 during air respiration was 86.0 +/- 6.2 Torr, and the correlation to arterial PO2 (Pao2) was 0.96 at normal blood flow. The arterial inflow was intermittently reduced in 10-15% stages of effective perfusion pressure (Peff). There was a hyperbolic decrease in PO2 when CBF was restricted in stages. A linear dependence between Peff, HP, and DF was found, which means that there is no autoregulation in the capillary bed at 45 degrees C. Transcutaneous PO2 can be also taken as an indication of CBF. The transcutaneous index, transcutaneous PO2/Pao2, is helpful for estimating local O2 availability.     

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.     

Electrooxidation of peroxynitrite in simulated body fluid at platinum electrode.

Peroxynitrite, a potent albeit deleterious biological oxidant, was electrooxidized at a bare platinum electrode in a medium of simulated body fluid (SBF) to nitrite anion and molecular oxygen. The SBF, prepared in accordance with literature procedure, has a pH of 7.4 and an ionic strength of 0.16. In this medium, peroxynitrite was observed to undergo a diffusion-controlled (D=1.63 x 10(-5) cm2/s) oxidation process at a bare platinum electrode with a heterogeneous rate constant, k(s), of 0.029 cm/sec. The formal redox potential, Eo', of this biological oxidant was determined as 1.18 V with respect to saturated calomel electrode (SCE) in a 2e- oxidation process. The electrochemical response of the oxidation process, measured in unit of current, is observed to be concentration dependent with a limit of detection of 1.6 x 10(-7) M. The linear dynamic range of the observed current-concentration plot extends over the entire concentration range studied. These observations make this electrochemical method a feasible technique for quantitative determination of peroxynitrite in vivo and in vitro.     

Effect of transpulmonary and driving pressures on collateral gas flow in dog lungs

The effect of changing segment pressure (Ps) and airway opening pressure (Pao) on flow through a collaterally ventilating lung segment was evaluated in intact and excised dog lungs. He, N2, and SF6 were passed through the lung segment distal to a catheter wedged in a peripheral airway at driving pressures (Ps - Pao) between 0.25 and 2 cm H2O. Eight excised caudal lobes were studied at Pao = 5, 10, and 15 cm H2O. Flow was directly related to Ps - Pao and Pao and inversely related to the density of the gas. A dimensionless plot of the driving pressure normalized to a reference dynamic pressure as a function of Reynolds number (Re) indicated that flow through the segment behaved as if it were laminar at Re less than 100 and that increasing Pao increased the dimension of the pathways conducting flow as shown previously. Small changes in Ps had no effect on pathway geometry or on the pattern of flow through the segment at Pao = 10 and 15 cmH2O. At Pao = 5 cm H2O increasing segment pressure appeared to increase the dimensions of the flow pathways slightly. Similar changes in Ps - Pao had no consistent effect on flow pattern or pathway geometry in six anesthetized, paralyzed, vagotomized dogs at functional residual capacity or after widely opening the chest (Pao = 5 cm H2O). These results suggest that, at large lobe volumes, airways (including collateral pathways) are maximally dilated and therefore relatively insensitive to small changes in segment pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

Mechanical properties of small airways in excised pony lungs.

We evaluated the pressure-flow relationships in collaterally ventilating segments of excised pony lungs by infusing N2, He, Ne, or SF6 at known flows (V) through a catheter wedged in a peripheral airway. Measurements were made at segment- (Ps) to-airway opening (Pao) pressure differentials of 3-15 cmH2O when the lungs were held at transpulmonary pressures of 5, 10, and 15 cmH2O. The data were analyzed both by calculating collateral resistance (Ps-Pao/V) and by constructing Moody-type plots of normalized pressure drop [(Ps-Pao)/(1/2 rho U2, where rho is density and U is velocity)] against Reynolds number to assess the pattern of flow through the segment and the change in dimension of the flow channels as Ps and Pao were changed. The interpretations from these analyses were compared with radiographic measurements of the diameters of small airways within the collaterally ventilating lung segment at similar pressures. Collateral resistance increased as Ps-Pao increased at high Reynolds numbers, i.e., high flows or dense gas (SF6). Analysis of the Moody-type plots revealed that flow was density dependent at Reynolds number greater than 100, which frequently occurred when N2 was the inflow gas. The radiographic data revealed that small airway diameter increased as Ps-Pao increased at all lung volumes. In addition, at 5 cmH2O Pao, small-airway diameter was smaller for a given Ps in the nonhomogeneous case (Ps greater than Pao) than small-airway diameter for the same Ps in the homogeneous case (Ps = Pao). We interpret these data to suggest that the surrounding lung prevented the segment from expanding in the nonhomogeneous case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a regional increase in alveolar pressure on pulmonary blood flow.

We examined whether wedging a catheter (0.5 cm OD) into a subsegmental airway in dog (n = 6) or pig lungs (n = 5) and increasing pressure in the distal lung segment affected pulmonary blood flow. Dogs and pigs were anesthetized and studied in the prone position. Pulmonary blood flow was measured by injecting radiolabeled microspheres (15 microns diam) into the right atrium when airway pressure (Pao) was 0 cmH2O and pressure in the segment distal to the wedged catheter (Ps) was 0, 5, or 15 cmH2O and when Pao = Ps = 15 cmH2O. The lungs were excised, air-dried, and sectioned. Blood flow per gram dry weight normalized to cardiac output to the right or left lung, as appropriate, was calculated for the test segment, a control segment in the opposite lung corresponding anatomically to the test segment, the remainder of the lung containing the test segment (test lung), and the remainder of the lung containing the control segment (control lung). The presence of the catheter reduced blood flow in the test segment compared with that in the control segment and in the test lung. Blood flow was not affected by increasing pressure in the test segment. We conclude that, in studies designed to measure collateral ventilation in dog lungs, the presence of the wedged catheter is likely to have a greater effect on blood flow than the increase in pressure associated with measuring collateral airway resistance.     

Effect of flow direction on collateral ventilation in excised dog lung lobes

We determined the effect of flow direction on the relationship between driving pressure and gas flow through a collaterally ventilating lung segment in excised cranial and caudal dog lung lobes. He, N2, and SF6 were passed through the lung segment distal to a catheter wedged in a peripheral airway. Gases were pushed through the segment by raising segment pressure (Ps) relative to airway opening pressure (Pao) and pulled from the segment by ventilating the lobe with the test gas, then lowering Ps relative to Pao. Driving pressures (Ps - Pao) between 0.25 and 2 cmH2O were evaluated at Pao values of 5, 10, and 15 cmH2O. Results were similar in cranial and caudal lobes. Flow increased as Ps - Pao increased and was greatest at Pao = 15 cmH2O for the least-dense gas (He). Although flow direction was not a significant first-order effect, there was significant interaction between volume, driving pressure, and flow direction. Dimensional analysis suggested that, although flow direction had no effect at Pao = 10 and 15 cmH2O, at Pao = 5 cmH2O, raising Ps relative to Pao increased the characteristic dimension of the flow pathways, and reducing Ps relative to Pao reduced the dimension. These data suggest that at large lobe volumes, airways (including collateral pathways) within the segment are maximally dilated and the stiffness of the parenchyma prevents any significant distortion when Ps is altered. At low lobe volumes, these pathways are affected by changes in transmural pressure due to the increased airway and parenchymal compliance.     

Calibration in dogs of a subcutaneous miniaturized glucose sensor using a glucose meter for blood glucose determination.

The feasibility of calibrating a glucose sensor by using a wearable glucose meter for blood glucose determination and moderate variations of blood glucose concentration was assessed. Six miniaturized glucose sensors were implanted in the subcutaneous tissue of conscious dogs, and the parameters used for the in vivo calibration of the sensor (sensitivity coefficient and extrapolated current in the absence of glucose) were determined from values of blood glucose and sensor response obtained during glucose infusion. (1) Venous plasma glucose level and venous total blood glucose level were measured simultaneously on the same sample, using a Beckman analyser and a Glucometer II, respectively. The regression between plasma glucose (x) and whole blood glucose (y) was y = 1.12x-0.08 mM (n = 114 values, r = 0.96, p = 0.0001). The error grid analysis indicated that the use of a Glucometer II for blood glucose determination was appropriate in dogs. (2) The in vivo sensitivity coefficients were 0.57 +/- 0.11 nA mM-1 when determined from plasma glucose, and 0.51 +/- 0.07 nA mM-1 when determined from whole blood glucose (t = 1.53, p = 0.18, n.s.). The background currents were 0.88 +/- 0.57 nA when determined from plasma glucose, and 0.63 +/- 0.77 nA when determined from whole blood glucose (t = 0.82, p = 0.45, n.s.). (3) The regression equation of the estimation of the subcutaneous glucose level obtained from the two methods was y = 1.04x + 0.56 mM (n = 171 values, r = 0.98, p = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

A technique to continuously measure arteriovenous oxygen content difference and P50 in vivo

In hypoxemic high-altitude polycythemic natives whose arterial O2 saturation (SaO2) normally ranges between 70 and 80%, three polyurethane catheters with both optical and polarographic sensors were inserted into the radial artery to measure SaO2 and O2 tension (PaO2), and three thermodilution fiber-optic balloon-tipped catheters were floated into the pulmonary artery to measure mixed venous O2 saturation (SvO2). Correlation of the in vivo SaO2, PaO2, and SvO2 values with the in vitro measurements was high (r = 0.97, 0.99, and 0.98, respectively). Both catheters were inserted in one polycythemic subject before and 4 days after isovolemic hemodilution. Data from the sensors were used to calculate arteriovenous O2 content difference (CaO2 - CvO2) and the O2 half-saturation pressure of hemoglobin (P50). The mean +/- 1 SD of the in vivo and in vitro P50 calculated with the Hill equation was 27.61 +/- 2.15 Torr and 27.35 +/- 1.60 Torr, respectively. The mean +/- 1 SD of the absolute difference between the in vivo and in vitro measurements was 1.16 +/- 1.21 Torr. The in vivo CaO2 - CvO2 correlated well with the in vitro measurements (r = 0.93), and the mean +/- 1 SD of the error in the catheter CaO2 - CvO2 measurements was 0.47 +/- 0.50 ml/dl. This technique appears to provide a useful measurement of blood gas exchange parameters and should be applicable to the study of exercise physiology and clinical regulation of O2 transport.     

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

Oxygen monitoring in neonatal medicine

In perinatal intensive care medicine, hypoxia and hyperoxia are both detrimental to the patient. PO2 measurements of arterial blood can be done in different ways: (1) by analysing blood obtained from an arteriopuncture; (2) by analysing blood obtained from an indwelling arterial (usually umbilical) catheter; (3) by using an indwelling catheter with a built-in O2 electrode; (4) by analysing alveolar air for PO2; (5) by measuring cutaneous arterial PO2 transcutaneously. The common principle of all electrodes mentioned is the polarographic one. It appears that for the clinician, the transcutaneous electrode will become the method of choice in the future because of its non-invasiveness to the patient and because of its capability to provide a continuous PO2 record.     

Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO(2) (TM) emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO(2) emulsions with very fine emulsion particles (< 0.2 mum) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration-based oxygen diffusion coefficients has also been discussed.     

Length-tension relationship of the external anal sphincter muscle: implications for the anal canal function

The length at which a muscle operates in vivo (operational length) and the length at which it generates maximal force (optimal length) may be quite different. We studied active and passive length-tension characteristics of external anal sphincter (EAS) in vivo and in vitro to determine the optimal and operational length of rabbit EAS. For the in vitro studies, rings of EAS (n = 4) were prepared and studied in a muscle bath under isometric conditions. For in vivo studies, female rabbits (n = 19) were anesthetized and anal canal pressure was recorded by use of a sleeve sensor placed in the custom-designed catheter holders of 4.5-, 6-, and 9-mm diameters. Measurements were obtained at rest and during EAS electrical stimulation. Sarcomere length of EAS muscle was measured by laser diffraction technique with no probe and three probes in the anal canal. In vitro studies revealed 2,054 mN/cm(2) active tension at optimal length. In vivo studies revealed a probe size-dependent increase in anal canal pressure and tension. Maximal increase in anal canal tension with stimulation was recorded with the 9-mm probe. Increases in anal canal tension with increase in probe size were completely abolished by pancuronium bromide. EAS muscle sarcomere length without and with 9-mm probe in the anal canal were 2.11 +/- 0.08 and 2.99 +/- 0.07 microm, respectively. Optimal sarcomere length, based on the thin filament length measured by thin filament analysis, is 2.44 +/- 0.10 microm. These data show that the operational length of EAS is significantly shorter than its optimal length. Our findings provide insight into EAS function and we propose the possibility of increasing anal canal pressure by surgical manipulation of the EAS sarcomere length.     

温度、盐度对强壮箭虫耗氧率和窒息点的影响

研究了不同温度、盐度下强壮箭虫的耗氧率和窒息点.结果表明:温度和盐度均对强壮箭虫的耗氧率和比耗氧率有显著影响.试验温度在5℃-25℃,强壮箭虫个体耗氧率(IO)和比耗氧率(SO)开始随温度的升高而升高,之后呈明显下降趋势.其回归方程分别为y=0.0058x3 -0.2956x2+4.415x-8.7816(R2=0.99,P＜0.05)和y=0.0011x3 -0.0546x2+0.8161x-1.6232(R2=0.99,P＜0.05),数值分别为6.30～11.71μg·ind-1·h-1和1.22 ～2.16μg· mg-1·h-1,窒息点为4.18～6.87 mg·L-1.盐度10 ～40条件下,IO和SO随盐度的升高逐渐下降,回归方程分别为y=-0.0068x2 -0.1412x+21.702(R2=0.89,P＜0.05)和y=-0.0013x2-0.0261x+4.0114( R2 =0.89,P＜0.05),数值分别为4.98～17.73μg ·ind-1·h-1和0.92～3.56 μg·mg-1 ·h-1,窒息点为4.02～6.24 mg·L-1.对强壮箭虫与其他水生动物的耗氧率和窒息点进行比较得出,强壮箭虫是一种狭氧性浮游动物.     

A decrease in sister chromatid exchange frequency during the prolonged cultivation of human lymphocytes

Sister chromatid exchange (SCE) frequency at different times of fixation was studied in human lymphocyte cultures obtained from 6 donors. No differences were found in the SCE frequency between human lymphocyte cultures fixed at 72 and 96 hours of incubation (10.61 +/- 0.85 and 10.15 +/- 0.81 SCE per cell, respectively). However, a decreased SCE frequency (8.11 +/- 0.36 SCE per cell) was observed in cultures fixed at 120 hours of incubation. For a more detailed studies, one lymphocyte culture was fixed at different times of incubation (from 56 to 128 hours, at each a 8 hours). A slight increase in SCE frequencies was found at the interval between 56 and 88 hours of incubation, while starting from 104 hours of incubation a marked decrease in the SCE frequency was observed. Time-dependent changes in the SCE frequency may be described by the equation y = -1.8614 + 0.3922x - (2.5183 x 10(-3))x2, where y is the number of SCEs per cell, and x--the duration of culture incubation in hours. The observed phenomenon may be associated with changes in proportion of T and B lymphocytes, or with heterochromatization of chromosomes during a prolonged cultivation, or with an early in vitro stimulation of the in vivo long-lived lymphocytes that may be more damaged than the in vivo short-lived and the in vitro late-stimulating ones.     

Oxygen binding and its allosteric control in hemoglobin of the primitive branchiopod crustacean Triops cancriformis

Branchiopod crustaceans are endowed with extracellular, high-molecular-mass hemoglobins (Hbs), the functional and allosteric properties of which have largely remained obscure. The Hb of the phylogenetically ancient Triops cancriformis (Notostraca) revealed moderate oxygen affinity, cooperativity and pH dependence (Bohr effect) coefficients: P(50) = 13.3 mmHg, n(50) = 2.3, and Phi = -0.18, at 20 degrees C and pH 7.44 in Tris buffer. The in vivo hemolymph pH was 7.52. Bivalent cations increased oxygen affinity, Mg(2+) exerting a greater effect than Ca(2+). Analysis of cooperative oxygen binding in terms of the nested Monod-Wyman-Changeux (MWC) model revealed an allosteric unit of four oxygen-binding sites and functional coupling of two to three allosteric units. The predicted 2 x 4 and 3 x 4 nested structures are in accord with stoichiometric models of the quarternary structure. The allosteric control mechanism of protons comprises a left shift of the upper asymptote of extended Hill plots which is ascribable to the displacement of the equilibrium between (at least) two high-affinity (relaxed) states, similar to that found in extracellular annelid and pulmonate molluscan Hbs. Remarkably, Mg(2+) ions increased oxygen affinity solely by displacing the equilibrium between the tense and relaxed conformations towards the relaxed states, which accords with the original MWC concept, but appears to be unique among Hbs. This effect is distinctly different from those of ionic effectors (bivalent cations, protons and organic phosphates) on annelid, pulmonate and vertebrate Hbs, which involve changes in the oxygen affinity of the tense and/or relaxed conformations.     

Relation of transcutaneous to arterial pO2 in hypoxaemia, normoxaemia and hyperoxaemia. Investigations in adults with normal circulation and in patients with circulatory insufficiency

The transcutaneous oxygen tension was monitored continuously by a heated cutaneous polarographic electrode in 7 adult intensive care patients, 12 patients without circulatory insufficiency, and 5 healthy volunteers, Arterial pO2 values were varied from hypoxaemia to normoxaemia and hyperoxaemia by variations of the inspired oxygen concentration. In normal volunteers and in patients without circulatory failure, transcutaneous pO2 indicated on an average about 81-92% of the arterial pO2 in normoxaemia and hyperoxaemia with a correlation coefficient of 0.97. In hypoxaemia there was an over-proportional decrease of the transcutaneous pO2 to a mean value of 44% fo the arterial pO2. In one case the transcutaneous pO2 reproducibly dropped to zero at paO2 values of 41 respectively 38 mm Hg (5.5 respectively 5.1 kPa). In intensive care patients the transcutaneous pO2 values were considerably lower than the paO2 values. There was no constant transcutaneous to arterial pO2 ration in most of the intensive care patients at different pO2 levels. In adults without disturbance of peripheral perfusion paO2 can be predicted with satisfactory accuracy from transcutaneous pO2 values in normoxaemia and in hyperoxaemia. In hypoxaemia and in circulatory insufficiency, the transcutaneous pO2 is only an indicator of the trend of the arterial pO2. Under these conditions it does not allow a quantitative estimate of paO2 changes.     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.