1-Monoglyceride production from lipase-catalyzed esterification of glycerol and fatty acid in reverse micelles

Glycerol-fatty acid esterification has been conducted with lipase from R. delemar in water/AOT/isooctane reverse micellar media, with the major product being 1-monoglyceride, a useful food-emulsifier. 1,3-diglyceride was also synthesized, but to a much lesser extent. For a given set of initial conditions, the reaction productivity, measured in terms of the initial product formation rate, V(0), and the final or equilibrium concentration of product, is optimal for a particular concentration of each surfactant, fatty acid, glycerol, and water. Many of these optimal values correlate well with a "critical" region on the phase diagram. Also, results indicate lipase-catalyzed esterification stops due to the achievement of kinetic equilibrium expect for a few cases where enzyme deactivation is severe. Dynamic light scattering was employed to examine the influence of water, glycerol, and fatty acid on micellar and interfacial structure. Results from this technique indicate enzyme kinetic are linked to interfacial phenomena and the presence of substrates at the interfacial region.     

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.     

Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.     

Effects of fasting on plasma lipids and cholesterol esterification in plasma, liver and intestinal mucosa in the char (Salmo alpinus L.)

1. CoA-dependent cholesterol esterification, measured as esterification of 3H-cholesterol, was demonstrated in homogenates of liver and intestinal mucosa of the char (Salmo alpinus L.). 2. Plasma concentration of triacylglycerols, unesterified and total cholesterol were significantly reduced to 43, 58 and 72% of the control values, respectively, after 6 weeks fasting. 3. The rate of cholesterol esterification in plasma and liver homogenate was significantly lower in the fasted fish compared to the controls, but the esterification activity in the homogenate of intestinal mucosa increased twofold in the fish fasted for 6 weeks.     

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of Receptor Induced Changes in Intracellular Free Calcium in Human Platelets

Abstract

The intracellular free calcium concentration plays a key role as second messenger in the regulation of cellular reactions. Post-receptor events can be investigated by measurement of free calcium concentration in cells. Our experience in measuring the intracellular free calcium concentration in platelets with the use of the fluorescent indicator quin-2-tetraacetoxymethyl- ester is described. Possible pitfalls in the preparation procedures of the platelets are discussed as well as critical steps and the limitations of the quin-2-method. The methodological approach is demonstrated by the presentation of an investigation with the adenylate cyclase inhibitors adenosine-5′-diphosphate and epinephrine as well as their interrelationship. The potentiation effect of epinephrine to adenosine 5′-diphosphate on platelet function such as aggregation is accompanied by a potentiation of the effect of these two platelet activators in elevating the intracellular free calcium concentration. Adenosine 5′-diphosphate elevates intra-platelet free calcium alone, whereas epinephrine acting through stimulation of the alpha₂-receptor needs another permissive factor to immediately elevate the intra-platelet free calcium.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Biphasic stimulation of cellular calcium concentration by 3,5,3'-triiodothyronine in rat thymocytes

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria

Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extramitochondrial free calcium, and finally activation of the permeability transition. Relationships between these phases, free and total matrix calcium concentration, and phosphate concentration are investigated in rat liver and brain mitochondria. Slow, continuous calcium infusion is employed to avoid transient bioenergetic consequences of bolus additions. Liver and brain mitochondria undergo permeability transitions at precise matrix calcium loads that are independent of infusion rate. Cytochrome c release precedes the permeability transition. Cyclosporin A enhances the loading capacity in the presence or absence of acetoacetate. A remarkably constant free matrix calcium concentration, in the range 1-5 microM as monitored by matrix-loaded fura2-FF, was observed when total matrix calcium was increased from 10 to at least 500 nmol of calcium/mg of protein. Increasing phosphate decreased both the free matrix calcium and the matrix calcium-loading capacity. Thus the permeability transition is not triggered by a critical matrix free calcium concentration. The rate of hydrogen peroxide detection by Amplex Red decreased during calcium infusion arguing against a role for oxidative stress in permeability pore activation in this model. A transition between a variable and buffered matrix free calcium concentration occurred at 10 nmol of total matrix calcium/mg protein. The solubility product of amorphous Ca3(PO4)2 is consistent with the observed matrix free calcium concentration, and the matrix pH is proposed to play the major role in maintaining the low matrix free calcium concentration.     

Clinical validation of dialysable calcium in relation to other methods of serum calcium measurement.

Dialysable calcium (CaD) values were measured by a simple technique not interfered with by protein bound calcium and validation attempted by comparison with concentrations of ionised calcium (CaI) and clinical categorisation. CaD values were also compared with total calcium (CaT) and albumin adjusted calcium (CaA) concentrations. The normal ranges for CaD, CaT, CaA, and CaI were calculated from the results in healthy blood donors. In 50 normal subjects CaD was more highly correlated with CaI than CaT or CaA. The effects of lying down and of venous stasis in 10 normal subjects showed that CaD was slightly influenced by posture only, whereas CaT was noticeably affected by posture and venous stasis; CaA reduced but did not abolish these effects on CaT. The correlation coefficient of CaD and CaI in patients with chronic renal failure was 0.81. CaD was compared with CaT and CaA values in 293 consecutive hospital patients; discrepant results were obtained in 14.3% and 13.0% of cases respectively and there were some clinical grounds for accepting CaD as correct in 86% and 74% of these cases. Measurement of CaD is a simple, reliable method for estimating accurately the calcium concentration free from biologically inactive protein bound calcium.     

Simulation of postsynaptic glutamate receptors reveals critical features of glutamatergic transmission

Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following high-frequency stimulation. They also provide a new tool to analyze the interactions between metabotropic and ionotropic glutamate receptors.     

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol⁻¹. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol⁻¹ at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h. Received: 12 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Effect of polyenoic phospholipid therapy on lecithin cholesterol acyltransferase activity in the human serum

The effect of polyenoic phospholipids on the concentration of serum lipids and the activity of lecithin cholesterol acyltransferase (LCAT, E.C. 2.3.1.43) was investigated in 18 patients with chronic glomerulonephritis accompanied by hyperlipaemia and reduced rate of cholesterol esterification in the plasma. The effects of therapy were evaluated immediately after a 2-month period of treatment and again after a 3-month drug free interval following termination of the therapy. An immediate effect of the treatment was reflected in a significant increase in the fractional esterification rate (FER % .h-1) and a marked reduction of the concentration of triglycerides (TG). Discontinuation of the drug resulted in the return of TG and FER values to the initial levels and in a rise of total (TCH) and unesterified cholesterol (UCH), HDL-cholesterol (HDL-TCH) and the molar esterification rate (MER mumol.1-1.h-1). The activity of LCAT estimated by radioassay in common and endogenous substrates varied in parallel.     

Optimization of extraction of high-ester pectin from passion fruit peel (Passiflora edulis flavicarpa) with citric acid by using response surface methodology

A central composite design was employed to optimize the extraction of pectin with citric acid. The independent variables were citric acid concentration (0.086–2.91% w/v) and extraction time (17–102 min). The combined effect of these variables on the degree of esterification was investigated. Results have shown that the generated regression models adequately explained the data variation and significantly represented the actual relationship between the independent variables and the responses. Besides that, the citric acid concentration was the most important factor to affect the degree of esterification, as it exerted a significant influence on the dependent variable. Lower citric acid concentration increased the pectin degree of esterification. The surface response showed the relationships between the independent variables, and thus responses were generated. Through this surface, the satisfactory condition of 0.086% w/v citric acid for 60 min was established for extraction of high-ester yellow passion fruit pectin.     

Resolution of (R,S)‐ibuprofen catalyzed by immobilized Novozym40086 in organic phase

The enantioselective esterification of ibuprofen catalyzed by Novozym40086 was successfully conducted in organic solvent. Removing‐water reagent was added into the reaction mixture to remove water produced in the esterification. The effects of temperature, n‐hexanol concentration, ibuprofen concentration, and loading of enzymes were investigated. Under the condition of equilibrium, the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity (E) of 8.512 were obtained. The esterification reaction achieved its equilibrium in approximately 30 hours with conversion of 56% and ee_S of 93.78%. The predicted values of X and ee_S were 67.90% and 95.60%, respectively. The experimental value is approximately equal to the theoretical value, which indicates the feasibility of ideal models.     

The ecophysiological significance of calcium bicarbonate in the urine of subterranean rodents: testing a hypothesis

1. A comparative study of calcium and bicarbonate in the urine was carried out on the subterranean mole rat Cryptomys hottenttus and the terrestrial vlei rat Otomys irroratus. 2. The two species were kept on two different diets; carrots, a high calcium diet (41 mg/ 100 kg) or potatoes, a low calcium diet (14 mg/ 100g). 3. The results show that the urine of the mole rat contained high values of calcium bicarbonate on either diet. 4. The urine of the vlei rat showed high values of calcium bicarbonate only when kept on the high calcium diet. 5. From these results we assume that in subterranean rodents excretion of calcium bicarbonate is an adaptive mechanism to unload CO2 without increasing its concentration in the hypercapnic environment.     

Responsiveness of κ-carrageenan microgels to cationic surfactants and neutral salts

The objective of this study was to examine the responsiveness of chemically cross-linked κ-carrageenan microspheres to different types of neutral salt electrolytes as well as to surfactants of varying chain lengths. In the presence of increasing salt concentration microsphere size changed radically from D[4,3] values of 320 μm to approximately 160 μm. The level of salt concentration needed to bring about this change varied depending on electrolyte type. This common behaviour was attributed to the difference in free cationic counter-ions concentration between the inside and outside of the microsphere and can be explained due to the effect of the Donnan equilibrium. The rheological properties of these microgels in their swollen and collapsed states were also explored with results showing that the collapsed microspheres had a greater impact on the viscosity of the system probably as a result of some aggregation of the collapsed microgels at rest due to surface charge screening at these high salt concentrations. The effect of surfactant on microsphere size showed a dramatic drop in D[4,3] values from 320 μm to approximately 120 μm for BAC, DoTAB, MTAB and CTAB at specific critical concentrations. This critical aggregation concentration was found to increase linearly on a log–log scale with the critical micelle concentration of these surfactants in water, indicating that the alkyl chain length of the surfactants had an effect on the critical aggregation concentration.     

Esterification Rates of Main Organic Acids in Wines

The esterification rates (ratio of the concentration of an acid in the neutral ethyl ester form to total concentration of the acid) of main organic acids in wines were determined to study the extent of ethyl ester formation of organic acids. The esterification rates ranged from zero to 24.6%. The averaged values of table wines were from 6 to 16% for acetic and lactic acid, from 0.3 to 3.6% for succinic and malic acid, and from zero to 0.1% for tartaric acid. Sherries had higher esterification rates, about 1.6 to 6 times larger, than table wines. It was found that storage time and temperature influence the formation of ethyl esters, and it was suggested that the aging period required for the ester equilibrium is about one year for acetic and lactic acid, and more than two years for succinic, malic and tartaric acid. The possibility and the procedure to control wine quality during the aging process were discussed.