Oxygen reaction vessels.

Transcutaneous oxygen electrodes were modified to be more suitable as a component of oxygen reaction vessels. The temperature control system was removed from the transcutaneous electrode to decrease the thickness and improve the stability. The temperature control system was incorporated in the metal sleeve surrounding the glass reaction vessel to shorten the distance between the magnetic stirrer and stirring bar, enabling smooth stirring with a short magnetic bar. With these modifications, we have succeeded in reducing the vessel volume to about 0.5 ml, or two to four times smaller than reaction vessels incorporating unmodified transcutaneous electrodes (vessel volume = 1-2 ml) and about twenty times smaller than reaction vessels using rod-shaped Clark electrodes (vessel volume about 10 ml). In another vessels modified as above, two optical guides were connected to the metal sleeve for irradiating the solution and receiving transmitted light simultaneously to enable simultaneous measurements of oxygen concentration absorption spectra. The relationship between oxygen concentration and absorption spectra of Hb is described as an application of this vessel.

Measurement of rat brain spectra.

A method of optically monitoring oxygen saturation of blood haemoglobin in the rat brain stem was developed. Optical fiber bundles were attached to the orifices of both ears of the rat to irradiate incident light from one ear and receive transmitted light from the other ear. Absorption spectra were measured using a white-light source and a photodiode array spectrophotometer. Stable measurements of optical time courses and absorption spectra were made using this "ear-to-ear" path. Oxygen saturation levels were calculated from spectra by using a suitable reference and an improved method of determining spectral peak heights.

Hypoxemia and hemodialysis-induced symptomatic hypotension.

We studied a possible involvement of hypoxemia in the pathophysiology of hemodialysis (HD)-induced symptomatic hypotension (SH). SH frequently recurred in 16 of 33 patients undergoing routine HD. These 16 patients showed a significantly (p less than 0.001) lower arterial oxygen tension (PaO2) prior to the start of HD than the remaining 17 patients without SH. This was the case when 10 patients with SH who had no additive risk factors for SH such as older age, excessive fluid loss and autonomic neuropathy were compared with a corresponding 11 patients without SH (p less than 0.001). Sequential monitoring of PaO2 throughout HD using the intravascular O2 electrode revealed that SH was never associated with particular changes in PaO2. The pattern of the decrease in, and the minimum value for, PaO2 during HD was not significantly different between the patients with and without SH. Furthermore, O2 administration during HD failed to prevent the occurrence of SH. These data suggest that pre-existing, but not HD-induced, hypoxemia is profoundly related to the pathophysiology of SH.

An assay method for cyclic AMP using high-performance liquid chromatography with pretreatment by alkaline phosphatase.

A convenient method for determination of cyclic AMP is described. This nucleotide was determined using high-performance liquid chromatography after the treatment of tissue extracts by alkaline phosphatase. Tissue extracts contain large amounts of materials which interfere with the measurement of cyclic AMP on the chromatogram. By the alkaline phosphatase treatment, these materials were completely converted to compounds which no longer interfered with the measurement. This method enables the detection of 2 pmol cyclic AMP, and is applicable to various tissue extracts which contain at least 0.1 nmol cyclic AMP/g wet wt.

Cultivation of Bacteroides fragilis in tissue culture.

The capacity of animal cells to support the growth of obligate anaerobes was shown by inoculating Bacteroides fragilis and Bacteroides thetaiotaomicron into cultures of Vero, HeLa, Chinese hamster ovary, and Chinese hamster lung cells. From an extremely small inoculum (nearly 1 cfu), B. fragilis multiplied up to 10(8) cfu/ml during aerobic cultivation in L-15 medium containing kanamycin sulfate (50 micrograms/ml) and 5% fetal bovine serum (total depth, 3-5 mm). Infecting bacteria formed a cluster on the cell monolayer and permitted the concomitant growth of infected Vero cells. Treatment of Vero cells with an inhibitor of respiration (sodium azide) arrested the bacterial growth reversibly. In contrast, an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, enhanced the growth of B.fragilis. By use of the thin oxygen electrode, low oxygen pressure was detected in the vicinity of the cultured Vero cells. Thus, respirating animal cells seem to provide a microenvironment with low enough oxygen pressure for the growth of some anaerobes.

Glucose tolerance and respiratory activities of human liver biopsies: their interdependence.

To determine a possible relation of hepatic oxidative activity to glucose metabolism, the rates of oxygen consumption of liver slices from patients with chronic liver diseases were polarographically measured. The livers from patients with chronic (persistent and aggressive) hepatitis and with normal glucose tolerance showed almost the same respiratory activity as those from patients with normal livers and normal glucose tolerance, whereas the livers from patients with chronic hepatitis and with diabetic glucose tolerance (ie, diabetes mellitus secondary to chronic hepatitis) showed only a half the normal level. The decreased rate of respiration was also observed in liver slices from cirrhotics with glucose intolerance. The decrease in respiration was found in patients with normal or hyperinsulinemia as well as hypoinsulinemia responding to oral glucose load. No liver tests so far examined, except the oral glucose tolerance test, correlated with hepatic respiratory activity. It is concluded that in patients with chronic liver diseases the defect of liver respiration has a close relation to the glucose metabolism and is not necessarily associated with histological change of the liver.

Growth of Bacteroides fragilis in rabbit tracheal organ culture: anaerobiosis and tissue respiration.

Rabbit tracheal explants supporting growth of inoculated Bacteroides fragilis in air were shown to keep low oxygen tension. Treating the explants with sodium azide induced high oxygen tension and arrested reversibly the growth of B. fragilis.

Hepatic aminopyrine N-demethylase system: interaction of aminopyrine with microsomal cytochrome P-450.

Interaction of aminopyrine with microsomal membrane-bound cytochrome P-450 was studied spectrophotometrically at various pH. Aminopyrine-induced type I spectral change in untreated rat microsomes was observed in neutral and alkaline media, and the absorption magnitude between peak and trough in the spectra increased markedly by increasing pH. On the other hand, an anomalous spectral change (lambda max, 425 nm; lambda min, 410 nm) was obtained in acid medium, and the absorption magnitude of the anomalous spectral change was enhanced by decreasing pH. The spectral dissociation constant for the anomalous aminopyrine-binding reaction at pH 6.32 was about one order of magnitude greater than that for the type I binding reaction at pH 8.22. The type of aminopyrine-induced spectral change differed depending upon the age and pretreatment of animals. Neonatal microsomes elicited only the anomalous spectral change in all pH media. Liver microsomes from 3-methylcholanthrene-pretreated rats showed a reverse type I spectral change. Antipyrine produced only a reverse type I spectral change in all microsomes tested, and the absorption magnitude was enhanced by decreasing the pH. In the presence of a saturated concentration of a reverse type I compound, i.e., ethanol or antipyrine, aminopyrine induced the type I spectral change, even in acid medium. The binding mechanism of cytochrome P-450 with aminopyrine is discussed on the basis of these results.

pH-dependent interaction of microsomal cytochrome P-450 with substrates. I. Effect of pH upon the interaction of exogenous substrates with membrane-bound cytochrome P-450.

The interaction of microsomal membrane-bound cytochrome P-450 with substrates was studied spectrophotometrically at various pH-values. The binding of type I compounds, hexobarbital and androstanedione, with cytochrome P-450, as determined by the magnitude of the type I spectral change of microsomes, was markedly enhanced at alkaline pH compared to that at acid pH. The pH-dependent spectral change could be reversed by changing the pH. The maximum absorption change (delta Amax) increased with increasing the pH, while the spectral dissociation constant (Ks) decreased. A similar pH-dependent binding reaction was also observed using a non-dissociative type I compound, cyclohexane. On the contrary, the absorbance magnitude between peak and trough in the aniline- or alcohol-induced difference spectrum of microsomes was enhanced by decreasing the pH, indicating easy complex formation of type II and reverse type I compounds with cytochrome P-450 in the acid rather than the alkaline region.

Intravascular oxygen monitoring with a polarographic oxygen cathode.

Thin flexible oxygen cathodes coated with heparin-dispersed cellulose diacetate were prepared for an intravascular monitoring of blood pO2. The effect of the thickness of cellulose diacetate on various characteristics of the cathode, such as its sensitivity, response, residual current, moving artefact, linearity, and protection against poisoning, were measured. Coating with six to ten layers of 7.5% cellulose diacetate resulted in a high level of protection for cathode against poisoning by blood constituents, while still leaving a sufficiently rapid response. An instillation system using heparinized saline has been designed for further prevention of local blood coagulation. At the same time this system maintains a stable conductance of the salt bridge and furthermore, enables in vivo calibration of the cathode sensitivity by supplying an instillation solution of a known oxygen tension. Using this electrode system, various intravascular pO2 measurements have been carried out, and one representative result is shown. The advantages and disadvantages of this type of separated electrode system compared with the combined type electrode are also discussed in detail.

Hemoperfusion, rate of oxygen consumption and redox levels of mitochondrial cytochrome c (+c1) in liver in situ of anesthetized rat measured by reflectance spectrophotometry.

Utilizing reflectance spectrophotometry, hemoperfusion, rate of oxygen consumption and redox level of mitochondrial cytochrome c (+c1) in livers in situ of anesthetized rats were measured. The transition to the anoxic state was induced by raising the pressure on the liver surface to more than the hepatic blood pressure by pressing with the tip of the optical guide of the reflectance spectrophotometer. During this transition, the average oxygen saturation of hemoglobin in the liver in situ decreased linearly with time until it became 10--20% of the total. This was followed by reduction of mitochondrial cytochrome c (+c1), which reached completion in 10--20 s. The measured O2 consumption rate remained constant until the percentage of oxyhemoglobin in situ decreased to a critical level. There was then a decrease in the rate of O2 consumption which was accompanied by a progressive reduction of cytochrome c (+c1). It was shown that amounts of hemoglobin and mitochondrial respiratory chain cytochromes in the liver in situ could be measured non-invasively and could provide important signals for vital cellular functions. The changes in hemoperfusion and rate of O2 consumption of the liver in situ following ethanol ingestion were also shown in rats, and are briefly discussed with respect to possible application of this method to study the pathophysiology of tissues.

Effect of ethanol on hemoperfusion and O2 sufficiency in livers in vivo.

Hemoperfusion and rate of O2 uptake in the livers in vivo following ethanol ingestion were measured by reflectance spectrophotometry. Pressurization on the liver in situ above the sinusoidal blood pressure caused complete blocking of blood flow, followed by spectral changes due to transition from hepatic normoxia to anoxia. Analyses of such spectra provided informations as to the rate of O2 consumption in situ and the redox level of cytochrome c (+cl)) in the mitochondrial respiratory chain. The rate of O2 consumption thus calculated remained constant until the apparent O2-saturation of hemoglobin in situ decreased to less than 10% of the total of hemoglobin. In parallel with the decrease of the rate of O2 consumption, the apparent reduction level of cytochrome c (+Cl) increased. It was shown in fed rats that the ethanol ingestion (1 g/kg) stimulated O2 uptake in the liver by 29% which initially caused a decrease in O2-saturation of hemoglobin, followed by an increase in blood supply to the liver. Thus, the ethanol consumption resulted in an increase in hepatic oxidative metabolism, possibly leading to hepatic hypoxia and liver damage.

Change in the ultraviolet spectrum of solubilized Ca2+-dependent ATPase from sarcoplasmic reticulum due to binding with Ca2+ ions.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).