The effect of trans sodium crocetinate (TSC) in a rat oleic acid model of acute lung injury.

Trans sodium crocetinate is a novel drug, which has been shown previously to increase whole-body oxygen consumption during hemorrhagic shock. TSC has been suggested to work by increasing the diffusion rate of oxygen through plasma rather than on a specific symptom of hemorrhagic shock and has been suggested as a general treatment for hypoxemia. Thus, it might also be beneficial for treating respiratory insufficiencies. This study employed an oleic acid model of acute lung injury to determine if TSC could increase arterial PO2 in that model.

Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects.

The esterification of long-chain fatty acids in n-hexane catalyzed by nylon-immobilized lipase from Candida rugosa has been investigated. Butyl oleate (22 carbon atoms), oleyl butyrate (22 carbon atoms) and oleyl oleate (36 carbon atoms) were produced at maximum reaction rates of approximately equal to 60 mmol h(-1) g(-1) immobilized enzyme when the substrates were present in equimolar proportions at an initial concentration of 0.6 mol l(-1). The observed kinetic behavior of all the esterification reactions is found to follow a ping-pong bi-bi mechanism with competitive inhibition by both substrates. The effect of the chain-length of the fatty acids and the alcohols could be correlated to some mechanistic models, in accordance with the calculated kinetic parameters.

Trans-sodium crocetinate restores blood pressure, heart rate, and plasma lactate after hemorrhagic shock.

BACKGROUND: Trans-sodium crocetinate (TSC) has been shown to increase oxygen consumption during hemorrhagic shock. The current study was done to determine the effect of TSC on other parameters such as blood pressure, heart rate, blood pH, and lactate. METHODS: A rat model of hemorrhagic shock was used, in which a constant volume of blood is removed. RESULTS: TSC increased mean arterial blood pressure from a value (immediately after hemorrhage) of 35 mm Hg to a value of 75 mm Hg, and all treated animals survived. In contrast, blood pressure in control animals decreased, with most dying soon after the hemorrhage. TSC also lessened the tachycardia which resulted from the hemorrhage. Blood pH did not decrease as much when TSC was given, and plasma lactate levels were greatly reduced. CONCLUSION: It would appear that TSC is a promising initial treatment for hemorrhagic shock.

Asymmetric ketone reduction with immobilized yeast in hexane: biocatalyst deactivation and regeneration.

There is a need to develop methods for producing enantiomerically pure pharmaceuticals because the racemic mixtures made today will probably not be allowed in the future. Synthetic chiral catalysts are being developed for this purpose, as well as new product separation techniques. Another possible option is to use biocatalysts, such as purified enzymes or whole microbial cells, since these can result in the production of mostly a single enantiomer. This study emphasizes the use of alginate-entrapped yeast cells to catalyze the reduction of ketones as a model system. The emphasis is on the factors that might limit the reactivity of such cells, such as equilibrium conditions, substrate or product inhibition, solvent toxicity, loss of cell viability, or the degradation of intracellular levels of enzymes or cofactors. It was found that there was a progressive loss of catalytic activity of the immobilized yeast cells, which appeared to be mainly associated with a loss of cell viability and a decline of intracellular NAD(H) levels during the reaction. The other factors investigated did not have a large effect. A regeneration scheme was developed in order to replenish the intracellular NAD(H) lost during the reaction, which involved removing the biocatalyst from the reaction and supplying the cells with a nutrient source. This resulted in an increase in the NAD(H) to initial levels and also resulted in a maintenance of the ketone reduction rate over time.

Intravenous crocetinate prolongs survival in a rat model of lethal hypoxemia.

OBJECTIVE: To examine whether a carotenoid, trans-sodium crocetinate, has beneficial effects on hemodynamic status and short-term outcome in a rat model of lethal hypoxemia. DESIGN: Randomized, placebo-controlled study. SETTING: Medical school laboratory. SUBJECTS: Eighteen spontaneously breathing, anesthetized Sprague-Dawley rats (six per group). INTERVENTIONS: Rats underwent instrumentation to measure blood pressure, aortic and renal blood flow, arterial blood gases, bladder epithelial oxygen tension (by an intraluminal Clark electrode), and hepatic microvascular oxygen tension (measured by porphyrin phosphorescence). After stabilization, the rats were subjected to breathing 10% inspired oxygen concentration. After 10 mins, they were administered 1.25 mL/kg intravenous boluses of either isotonic saline (control), normal strength crocetinate (40 microg/mL), or a concentrated crocetinate solution (60 microg/mL). These boluses were repeated at 30-min intervals until either death or 3 hrs had elapsed. MEASUREMENTS AND MAIN RESULTS: With the onset of hypoxemia, we observed a rapid reduction in blood pressure and renal blood flow, maintenance of aortic blood flow, an increase in arterial base deficit, and falls in oxygen tensions in arterial blood, bladder epithelium, and hepatic microvasculature. A progressive deterioration in the control rats was noted, with only two of the six animals surviving for 3 hrs. However, all 12 rats in the two crocetinate groups survived for 3 hrs, with hemodynamic stability until 150 mins and a slow decline thereafter. CONCLUSIONS: Trans-sodium crocetinate improved hemodynamic status and prolonged survival in this model of severe acute hypoxic hypoxia. To our knowledge, this is the first demonstration of an intravenous agent having such an effect.

Effect of aeration during cell growth on ketone reactions by immobilized yeast.

The effect of aeration during cell growth on the subsequent reduction of 2-hexanone and 2-octanone by yeast cells entrapped in calcium alginate beads was studied. The reactions were conducted using 2-propanol as a sacrificial substrate to regenerate the cofactor NAD(H), and a mixture of (S)- and (R)-alcohols was produced. The use of strictly aerobic conditions when growing the cells resulted in the highest initial reaction rates, as well as the production of only a single product (i.e., the enantiomeric excess of the (S)-alcohols was 1.0). However, initial reaction rates decreased proportionally with fermentation time regardless of whether the yeast were grown aerobically or under both aerobic and anaerobic conditions. The data also suggest that it is the aerobic (or anaerobic) condition, rather than the cell growth phase, which is responsible for the results seen.

A novel fluid resuscitation therapy for hemorrhagic shock.

Fluid resuscitation is the usual therapy for hemorrhagic shock, and frequently consists of the infusion of large volumes of electrolyte solutions. However, to be successful, this therapy should be implemented soon after injury. A new treatment method in which the infusion could be delayed might result in a greater survival rate. Reducing the volume of fluid needed is also important. Both of these aspects of fluid resuscitation therapy were addressed in this study by supplementing the electrolyte solution with trans-sodium crocetinate (TSC). Rats were subjected to a severe hemorrhage, with 55% (or greater) of the estimated blood volume being removed over a period of approximately 10 min. There were five animals in each treatment group, and two types of experiments were done. In one, a bolus injection of TSC (or saline control) was given immediately after hemorrhage, followed 30 min later with an infusion of isotonic saline. In the other experiments, reduced infusion volumes of a TSC-saline infusion fluid were used. In both cases, TSC resulted in the survival of the animals while the controls all died. Whole-body oxygen consumption also increased with TSC, reaching 75% of the normal resting value after about 15 min. This correlates well with the increased survival rates seen, since mortality after hemorrhagic shock is associated with decreased oxygen consumption. These results suggest that the use of TSC could allow for later implementation of fluid resuscitation therapy as well as reducing the volume needed.

Adsorptive control of water in esterification with immobilized enzymes: I. Batch reactor behavior.

Reducing the influence of an undesired product in an enzymatic reaction could have a significant impact on the productivity of such systems. Here, we focus on the removal of water formed during an enzymatic esterification in a batch reactor. A commercial immobilized lipase preparation, known as Lipozyme, is used as the biocatalyst and propionic acid and isoamyl alcohol dissolved in hexane are the substrates. In this system, the water formed will partition between the catalyst and the medium. As the more polar reactants are converted into the less polar ester product, the water is partitioned more towards the biocatalyst and the accumulation of water eventually causes lower reaction rates. Addition of a strong-acid cation exchange resin in sodium form is found to control the water accumulation on the biocatalyst without stripping the essential water needed for the enzyme to function and substantial improvements in conversion are achieved. A mathematical model is developed to describe the batch reaction behavior with and without added absorbent, which successfully predicts the behavior of water and its effects.

Adsorptive control of water in esterification with immobilized enzymes: II. fixed-bed reactor behavior.

Experimental and theoretical studies are conducted to understand the dynamic behavior of a continuous-flow fixed-bed reactor in which an esterification is catalyzed by an immobilized enzyme in an organic solvent medium. The experimental system consists of a commercial immobilized lipase preparation known as Lipozyme as the biocatalyst, with propionic acid and isoamyl alcohol (dissolved in hexane) as the reaction substrates. A complex dynamic behavior is observed experimentally as a result of the simultaneous occurrence of reaction and adsorption phenomena. Both propionic acid and water are adsorbed by the biocatalyst resulting in lower reaction rates. In addition, an excessive accumulation of water in the reactor leads to a rapid irreversible inactivation of the enzyme. A model based on previously-obtained adsorption isotherms and kinetic expressions, as well as on adsorption rate measurements obtained in this work, is used to predict the concentration and thermodynamic activity of water along the reactor length. The model successfully predicts the dynamic behavior of the reactor and shows that a maximum thermodynamic activity of water occurs at a point at some distance from the reactor entrance. A cation exchange resin in sodium form, packed in the reactor as a selective water adsorbent together with the catalyst particles, is shown to be an effective means for preventing an excessive accumulation of water formed in the reaction. Its use results in longer cycle times and greater productivity. As predicted by the model, the experimental results show that the water adsorbed on the catalyst and on the ion exchange resin can be removed with isoamyl alcohol with no apparent loss in enzyme activity.

Synthesis of lovastatin with immobilized Candida rugosa lipase in organic solvents: Effects of reaction conditions on initial rates.

Lipase from Candida rugosa immobilized on a nylon support has been used to synthesize lovastatin, a drug which lowers serum cholesterol levels, by the regioselective acylation of a diol lactone precursor with 2-methylbutyric acid in mixtures of organic solvents. Analogs of lovastatin having a different side chain were also obtained through this method by reacting the diol substrate with different carboxylic acids. The selection of reaction conditions that maximize the initial reaction rate is investigated. Since the diol substrate has very low solubility in non-polar solvents, reaction solvents consisting of mixtures of hexane with a different, more polar cosolvent are considered. For each of the cosolvent mixtures studied, the reaction rate is maximum for an intermediate percentage of cosolvent in hexane. With total concentrations of the diol lactone in the range 6.25-12.5 mM, maximum initial rates correspond approximately to those cosolvent concentrations that permit a complete solubilization of the substrate. At higher cosolvent concentrations, lower rates are obtained. When considering the same dissolved substrate concentration, the reaction rate was found to increase with increasing values of logP(mix) and decreasing values of the dielectric constant, when varying the composition of a binary solvent mixture. However, when comparing different cosolvents, no general trend with respect to these properties was observed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56:671-680, 1997.

Fatty acid esterification using nylon-immobilized lipase.

The esterification of a long-chain fatty acid was conducted using a nylon-immobilized lipase from Candida cylindracea in a nearly anhydrous, nonpolar organic medium, hexane. Butyl laurate was produced from lauric acid and n-butanol at a maximum initial reaction rate of 37 mmol/h. g immobilized enzyme when the substrates were present in equimolar amounts at an initial concentration of 0.5 mol/L. Lower rates were obtained using nonstoichiometric amounts of the substrates. The rate of reaction increased with temperature, reaching a maximum between 35 and 45 degrees C and decreasing sharply at higher temperatures. (c) 1995 John Wiley & Sons, Inc.

Hemorrhagic shock in rats.

Rats are commonly used as models of hemorrhagic shock. Both constant-pressure and constant-volume protocols have been used, although the latter is considered by some to be a better model of human shock. However, differences in survival have been reported when the same experiment is done by different investigators. Changes in oxygen consumption have been reported to correlate with mortality after shock, and these changes may be implicated in the variability seen in different experimental studies. Using the constant-volume model, in which a given percentage of the animal's estimated blood volume is withdrawn, we have determined oxygen consumption rates before and after hemorrhage in Sprague Dawley rats. Our results indicate that specific oxygen consumption rates decrease once rats reach a certain weight, but removing a given percentage of blood reduces the rate by a constant amount regardless of the weight of the animal. In addition, there appears to be a "critical" level of oxygen consumption needed for survival. Thus use of a constant-volume protocol could result in survival for smaller but not larger rats. It is suggested that rat weights must be the same to compare results from different investigators.

Evaluating the oxygen diffusing capacity of maximally stimulated canine muscle.

A series of studies over the past several years has been concerned with O2 consumption in the gastrocnemius muscle of the dog. Those experiments indicate that the maximal O2 consumption may be diffusion limited and suggest that such data can be correlated using a parameter, the O2 diffusing capacity, that combines different effects. An analysis of those data is presented here with use of multiple linear regression techniques, which indicate that, even if the experimental conditions are different, the diffusing capacity can be quantitatively evaluated if the blood flow rate, hemoglobin content, and blood pressure are known. Although the data are for a specific tissue, similar results might be expected when O2 consumption is considered in other tissues.

The effect of crocetin on hemorrhagic shock in rats.

There is a reduction in oxygen consumption during hemorrhagic shock, and it has been suggested that this correlates with mortality. Recent data indicate that the consumption of oxygen may depend on its diffusion from the erythrocytes to the mitochondria; thus, enhancing this rate might increase tissue oxygen extraction during hypovolemia. Crocetin, a carotenoid compound which has been shown to increase oxygen diffusivity, was used in rats bled 40% of their blood volumes, and resulted in increased whole-body oxygen consumption and survival rates. Magnetic resonance spectroscopy data also indicate that crocetin increased oxygen uptake by muscle. Other factors which might account for these results, such as possible effects of crocetin on red cell deformability and mitochondrial respiration rates, were also investigated, but the mechanism of action seems to be related to the increased diffusion of oxygen through plasma.

Glucose effects on oxygen consumption by the arterial wall.

The effect of both D- and L-glucose on the rate of oxygen consumption in rat aorta was determined. Oxygen uptake (V02) was found to decrease when the concentration of D-glucose in the medium was increased, with the same effect being found when L-glucose, rather than D-glucose, was used. Based on these results, it would appear that the decrease in the vascular wall oxygen consumption which results from increased glucose levels is not due to a metabolic effect since one of the isomers (D) is metabolized and the other (L) is not. It is suggested, instead, that these results may be be due to a change in the diffusion of oxygen. Possible implications for atherogenesis are also discussed.

The effects of thermogenic agents on hindlimb oxygen consumption in the dog: ICI D7114 and noradrenaline.

The thermogenic action of beta-adrenoceptor agonists may be due, in part, to increased metabolism in skeletal muscle. Previous results suggest that vasoconstriction is also necessary, and that the effect can be blocked by vasodilators. Both noradrenaline and the beta-3 agonist, ICI D7114, were studied using two dog hindlimb protocols. During constant perfusion conditions, ICI D7114 caused a significant increase in hindlimb oxygen consumption although it is a vasodilator. Noradrenaline resulted in a smaller rise in oxygen consumption, and produced a marked vasoconstriction. Both noradrenaline and ICI D7114 resulted in decreased oxygen consumption when the blood flow was allowed to vary in response to the drug treatment. The results suggest that changes in tissue oxygen consumption caused by beta-agonists are not related to vasomotion.

Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors.

Direct esterifications using a nylon-immobilized lipase from Candida cylindracea were carried out in batch and continuous-flow reactors. The immobilized enzyme was effective in catalyzing the synthesis of ethylpropionate, isoamylpropionate, and isoamylbutyrate. With ethanol dissolved in hexane as a substrate, the maximum initial esterification rate was 0.02 mole/(h x g of immobilized protein), but the enzyme was stable only when the substrate concentrations were lower than 0.2 M. With isoamyl alcohol in hexane as a substrate, esterification rates as high as 0.085 mole/(h x g of immobilized protein) were observed and the immobilized enzyme was stable over a much broader concentration range. However, in this case, the use of a solvent, such as hexane, was not necessary for esterification, and the enzyme could be employed in equimolar acid/alcohol mixtures. A packed-bed reactor was operated successfully for the continuous synthesis of esters. The reactor was stable for long periods of time, and the steady-state performance could be accurately predicted on the basis of batch reaction experiments.

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.