Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases

The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated. COX-1 and COX-2 were able to minimally metabolize many of these species. However, the lipoamino acids were efficiently oxygenated by 12S- and 15S-LOs. The kinetics and products of oxygenation by LOs were characterized. Whereas 15S-LOs retained positional specificity of oxygenation with these novel substrates, platelet-type 12S-LO acted as a 12/15-LO. Fatty acid oxygenases may play an important role in the metabolic inactivation of lipoamino acids or vanilloids or may convert them to bioactive derivatives.     

Improvement of tumor oxygenation by mild hyperthermia

There is now abundant evidence that oxygenation in rodent, canine and human tumors is improved during and for up to 1-2 days after heating at mild temperatures. An increase in tumor blood perfusion along with a decline in the oxygen consumption rate appears to account for the improvement of tumor oxygenation by mild hyperthermia. The magnitude of the increase in tumor pO(2), determined with oxygen-sensitive microelectrodes, caused by mild hyperthermia is less than that caused by carbogen breathing. However, mild hyperthermia is far more effective than carbogen breathing in increasing the radiation response of experimental tumors, probably because mild hyperthermia oxygenates both (diffusion-limited) chronically hypoxic and (perfusion-limited) acutely hypoxic cells, whereas carbogen breathing oxygenates only the chronically hypoxic cells. Mild hyperthermia is also more effective than nicotinamide, which is known to oxygenate acutely hypoxic cells, in enhancing the radiation response of experimental tumors. The combination of mild hyperthermia with carbogen or nicotinamide is highly effective in reducing the hypoxic cell fraction in tumors and increasing the radiation response of experimental tumors. A primary rationale for the use of hyperthermia in combination with radiotherapy has been that hyperthermia is equally cytotoxic toward fully oxygenated and hypoxic cells and that it directly sensitizes both fully oxygenated and hypoxic cells to radiation. Such cytotoxicity and such a radiosensitizing effect may be expected to be significant when the tumor temperature is elevated to at least 42-43 degrees C. Unfortunately, it is often impossible to uniformly raise the temperature of human tumors to this level using the hyperthermia devices currently available. However, it is relatively easy to raise the temperature of human tumors into the range of 39-42 degrees C, which is a temperature that can improve tumor oxygenation for up to 1-2 days. The potential usefulness of mild hyperthermia to enhance the response of human tumors to radiotherapy by improving tumor oxygenation merits continued investigation.     

Characterization of the near infrared absorption spectra of cytochrome aa3 and haemoglobin for the non-invasive monitoring of cerebral oxygenation

Near infrared (IR) spectroscopy can give continuous, direct information about cerebral oxygenation in vivo by providing signals from oxygenated and deoxygenated haemoglobin and cytochrome aa3. Due to a lack of precise spectral information and uncertainties about optical path length it has previously been impossible to quantify the data. We have therefore obtained the cytochrome aa3 spectrum in vivo from the brains of rats after replacing the blood with a fluorocarbon substitute. Near infrared haemoglobin spectra were also obtained, at various oxygenation levels, from cuvette studies of lysed human red blood cells. Estimates of optical path length have been obtained. The data were used to construct an algorithm for calculating the changes in oxygenated and deoxygenated haemoglobin and oxygenated cytochrome aa3 in tissue from changes in near IR absorption.     

A numerical model for the oxygenation of blood in lung capillaries--effect of nth order one-step kinetics of oxygen uptake by haemoglobin

A numerical model is described for the oxygenation of blood in lung capillaries by considering the transport mechanisms of molecular diffusion, convection and the facilitated diffusion due to the presence of haemoglobin. In order to represent the oxygen dissociation curve accurately in the model, the nth order one-step kinetics of oxygen uptake by haemoglobin has been used. The resulting system of coupled, non-linear partial differential equations is solved numerically. It is shown that the blood is required to traverse a larger distance in the capillary before becoming fully oxygenated with nth order one-step kinetics in comparison to first-order one-step kinetics.     

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia

Summary The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60–70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1–3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitriteinduced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - RBC red blood cell     

The oxygenation of cholesterol esters by the reticulocyte lipoxygenase

The arachidonate 15-lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV-spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.     

Heterotropic effects of chloride on the ligation microstates of hemoglobin at constant water activity.

Dimer-tetramer assembly reactions of the 10 CN-met ligation microstates of hemoglobin (Hb) were analyzed as a function of NaCl concentration while maintaining constant water activity by the addition of compensating sucrose. The assembly free energy for fully ligated cyanomet Hb and for fully oxygenated Hb becomes less favorable by 1.8 kcal when [NaCl] is increased from 0.08 to 0.7 M, whereas that of unligated Hb is practically insensitive to changes in [NaCl]. Values of 1.6 and 0.3 mol chloride release were found for the assembly of fully ligated and deoxy Hb, respectively; i.e., a net release of 1.3 mol chloride is coupled to the ligation of tetramers for both oxygen and cyanomet ligation. The ligation-linked salt component at constant water activity was evaluated to be 1.0 mol for the full oxygenation of the Hb tetramer in agreement with the overall value previously reported. When the detailed salt linkages accompanying all 16 stepwise cyanomet ligation reactions were experimentally resolved, only two "chloride" effects were found. The first chloride effect correlates with the ligation steps, which create tertiary constraint, and the second effect is coupled to the six switchpoints of quaternary T-->R transition. The distribution of these chloride effects agrees closely with predictions of the "symmetry rule mechanism." The total chloride release for CN-met ligation is in good agreement with that for oxygenation. Free energy contributions to assembly and cooperativity arising from the osmotic effects of chloride were found to be small for all ligation species.     

Cyanosis by methemoglobinemia in tadpoles of Cochranella granulosa (Anura: Centrolenidae)

Tadpoles inhabit generally well oxygenated rivers and streams, nevertheless they were found in areas with limited oxygen availability inside the rivers. To assess this feature, I examined factors that influence centrolenid tadpole behaviour using Cochranella granulosa. The tadpoles were reared in well-oxygenated and hypoxic environments and their development, survivorship and growth were compared. The tadpoles in oxygenated water acquired a pale color, while tadpoles in hypoxic water grew faster and were bright red and more active. In the oxygenated water, the ammonium, which had its origin in the tadpoles' urine and feces, was oxidized to nitrate. In contrast, in the hypoxic treatment, the nitrogen compounds remained mainly as ammonium. Presumably, the nitrate in oxygenated water was secondarily reduced to nitrite inside the long intestine coils, because all symptoms in the tadpoles point to methemoglobinemia, which can occur when the nitrite passes through the intestine wall into the bloodstream, transforming the hemoglobin into methemoglobin. This could be checked by a blood test where the percentage of methemoglobin was 2.3% in the blood of tadpoles reared in hypoxic condition, while there was a 19.3% level of methemoglobin in the blood of tadpoles reared in oxygenated water. Together with the elevated content of methemoglobin, the growth of the tadpoles was delayed in oxygenated water, which had high nitrate content. The study about quantitative food-uptake showed that the tadpoles benefit more from the food in hypoxic water, although they spent there more energy moving around than the tadpoles living in oxygenated but nitrate-charged water.     

A kinetic description of dioxygen motion within alpha- and beta-subunits of human hemoglobin in the R-state: geminate and bimolecular stages of the oxygenation reaction

Laser flash photolysis technique is used to study human hemoglobin (HbA) oxygenation. Monomolecular geminate oxygenation of triliganded R-state HbA molecules is described by a function of three exponentials. Geminate oxygenation of the alpha-subunit within R-state HbA is characterized by two components with time constants of 0.14 and 1 ns, while geminate oxygenation of the beta-subunit within HbA is characterized by two components with time constants of 1 and approximately 30 ns. Bimolecular oxygenation of triliganded R-state HbA molecules is described by a biexponential law. Two observed rate constants are assigned to oxygenation of the alpha- and beta-subunit within HbA. The bimolecular association rate constants for O(2) rebinding with the alpha- and beta-subunit within triliganded R-state HbA are k(alpha) = 18.8 +/- 1.3 (microM x s)(-1) and k(beta) = 52 +/- 4 (microM x s)(-1), respectively. The apparent quantum yields of photodissociation of the beta- and alpha-subunit within completely oxygenated R-state HbA differ from each other by a factor of 3.6 and are equal to 0.041 +/- 0.004 and 0.0114 +/- 0.0012, respectively. The apparent quantum yield of photodissociation of completely oxygenated R-state HbA is equal to 0.026 +/- 0.003.     

A numerical model for studying the effect of plasma layer on the process of blood oxygenation in the pulmonary capillaries

A two layer model for the blood oxygenation in pulmonary capillaries is proposed. The model consists of a core of erythrocytes surrounded by a symmetrically placed plasma layer. The governing equations in the core describe the free molecular diffusion, convection, and facilitated diffusion due to the presence of haemoglobin. The corresponding equations in the plasma layer are based on the free molecular diffusion and the convective effect of the blood. According to the axial train model for the blood flow proposed by Whitmore (1967), the core will move with a uniform velocity whereas flow in the plasma layer will be fully developed. The resulting system of nonlinear partial differential equations is solved numerically. A fixed point iterative technique is used to deal with the nonlinearities. The distance traversed by the blood before getting fully oxygenated is computed. It is shown that the concentration of O2 increases continuously along the length of the capillary for a given ratio of core radius to capillary radius. It is found that the rate of oxygenation increases as the core to capillary ratio decreases. The equilibration length increases with a heterogeneous model in comparison to that in a homogeneous model. The effect of capillary diameters and core radii on the rate of oxygenation has also been examined.     

The function of hemocyanin in respiration of the blue crab Callinectes sapidus.

Blood PO2 in the blue crab Callinectes sapidus, a very active species of tropical origin, is lower at 22 degrees C than that of larger crabs in colder waters. These low oxygen levels permit its hemocyanin to be highly oxygenated at the gill, and to deliver almost half of its oxygen to the tissues in resting animals. Sustained muscular activity results in conspicuous decreases in blood PO2, pH and hemocyanin oxygenation. Although the venous reserve is fully utilized, hemocyanin oxygenation at the gill decreases so much that there is no change in its total quantitative function. The large Bohr shift becomes functional during activity, but its quantitative importance is not clear.     

Effect of differences in optical properties of intermediate oxygenated species of hemoglobin A0 on Adair constant determination

Careful evaluation of the so-called isosbestic properties of oxygenated and deoxygenated hemoglobin spectra demonstrates that the spectral changes are not strictly linear with respect to the degree of saturation. In order to quantify the extent of nonlinearity, optical measurements of O2 binding to human hemoglobin were made at different wavelengths in the Soret region approaching the presumed isosbestic point. The results indicate that the extinction coefficient of intermediate oxygenated hemoglobin is 1% less than that of the fully oxygenated hemoglobin, with a resulting 3% (+/- 0.15%) nonlinearity effect on measurements taken at the peak of the oxygenated hemoglobin spectrum (414 nm). The lack of isosbestic conditions allows one to investigate the functional properties of the oxygenated intermediates directly. The small difference in the absorbance of different oxygenated species has practically no influence on the determination of Adair constants at wavelengths removed from the critical isosbestic region.     

The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.     

Effect of oxygen binding on the dielectric properties of hemoglobin

The dielectric properties of horse hemoglobin have been investigated in the frequency range for 100 kcps to 15 Mcps at varying degrees of oxygenation. A linear dependence of the specific increment on the degree of oxygenation was found under a variety of experimental conditions, the increment of oxygenated hemoglobin being about 10% larger than that of deoxygenated hemoglobin. A similar difference was obtained with human adult and fetal hemoglobin. No variation of the dielectric parameters as reported by Takashima and Lumry could be detected.     

Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase

Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.     

Oxygenation of trans polyunsaturated fatty acids by lipoxygenase reveals steric features of the catalytic mechanism

Lipoxygenase, a nonheme iron dioxygenase, catalyzes the oxygenation of 1,4-diene units in polyunsaturated fatty acids, forming conjugated diene hydroperoxides as the primary products. The naturally occurring all-Z geometry for the olefins in the polyunsaturated fatty acid has long been thought to be a substrate requirement for the enzyme. A rigorous test of this hypothesis using the two isomeric (9E,12Z)- and (9Z,12E)-9,12-octadecadienoic acids was carried out. Both isomeric substrates were found to be catalytically oxygenated by soybean lipoxygenase 1 at a significant fraction of the rate of the reaction of the natural substrate, linoleic acid. Product determinations revealed that a thermodynamically unfavorable E to Z isomerization at the 9,10-position occurred when (9E,12Z)-9,12-octadecadienoic acid was converted into the 13-hydroperoxide by lipoxygenase 1. Determination of the stereochemistry at the oxygenated position in the products indicated that a comparable isomerization at the 12,13-position did not occur when the 9Z,12E isomer was employed. The distribution of products obtained from oxygenation at the 9-position supported the hypothesis that the enzyme catalyzes the reaction in one of two substrate orientations, conventional and head to tail reversed. The observations can be understood on the basis of the steric demands on intermediates in the proposed mechanism of action as well as by catalysis by the active-site iron atom.     

Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in Candida tropicalis cell suspensions.

The metabolism of glucose and xylose was studied as a function of oxygenation in suspensions of Candida tropicalis by 31P and 13C nuclear magnetic resonance spectroscopy. Both the rate of carbohydrate metabolism and the cytoplasmic pH were independent of the rate of oxygenation in cells metabolizing glucose. However, these two parameters were markedly dependent on the rate of oxygenation in C. tropicalis cells metabolizing xylose. For example, the cytoplasmic pH in fully oxygenated xylose-metabolizing cells was 7.8 but decreased to 6.3 in anoxic cells. In general, suspensions of cells consuming xylose had a lower rate of sugar uptake, a more acidic cytoplasmic pH, lower levels of sugarphosphomonoesters (SP) and ATP, higher levels of intracellular Pi, a more alkaline vacuolar pH, and a lower rate of extracellular Pi assimilation and polyphosphate synthesis than cells consuming glucose. These observations indicate that C. tropicalis metabolizing xylose is less energized than glucose-metabolizing cells. On both carbon sources, however, an inverse correlation between intracellular levels of SP and Pi was observed. Also, uptake of extracellular Pi correlated with the synthesis of polyphosphates within the cells. During anoxia, Pi was not taken up, and polyphosphates were hydrolyzed instead to fulfill the cells' requirements for phosphate.     

Oxygenation of phosphatidylcholine by human polymorphonuclear leukocyte 15-lipoxygenase

A cell-free human polymorphonuclear leukocyte preparation containing both 15- and 5-lipoxygenase activities was found to oxygenate phosphatidylcholine at carbon-15 of the arachidonic acid moiety. No oxygenation at carbon-5 was found. Under similar incubation conditions, soybean and rabbit reticulocyte 15-lipoxygenases also oxygenated phosphatidylcholine, whereas rat basophilic leukemia cell 5-lipoxygenase, rabbit platelet 12-lipoxygenase and rat liver cytochrome P-450 preparations did not. Our results suggest that the oxygenation of phospholipids may be a unique property of the 15-lipoxygenases.     

Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450

9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.     

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the maximum wavelength lambda max of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for lambda max of the fully oxygenated haemocyanin were 531.5 nm (pH less than 7.8) and 530.0 nm (pH greater than 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH less than 7.2) and 535.2 nm (pH greater than 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891-1895]. Only four amino acids of the four-hexamer were labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d. The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.