Lipid Emulsion In Rats Research Articles

Двадцать лет «серебряной пули» против интоксикации местными анестетиками

Местные анестетики (МА), как и все другие медикаменты, могут вызвать нежелательные явления. В отличие от крайне редких (приблизительно 1 : 1 000 000) аллергических реакций на МА интоксикация местными анестетиками (ИМА) значительно более вероятна (инцидентность приблизительно 1 : 1000), особенно с относительно высокими дозами, применяемыми для эпидуральных или больших блокад нервов. Лечение тяжелой ИМА от современных анестетиков мощного и длительного действия (например, бупивакаины) было очень трудным и почти безнадежным до успешного «липидного спасения» довольно высокими внутривенными дозами липидной эмульсии у крыс в 1998 году, затем у пациентов в 2006 году. Этот революционный метод (20% липидная эмульсия болюсом 100 мл в течение 2–3 минут, затем инфузией 200–250 мл в течение 15–20 хвилин, если не восстановится стабильность гемодинамики — рассмотреть повторный болюс или ускорение инфузии), образно названный «серебряной пулей» против ИМА, сначала рекомендовался лишь как средство отчаяния в случае безуспешной реанимации. Однако рекомендации Американского общества региональной анестезии 2010, 2012 и 2017 годов последовательно становились все менее консервативными, и теперь «липидное спасение» рекомендуется при первых признаках серьезной ИМА, сразу после обеспечения проходимости дыхательных путей. Вывод: один литр 20% липидной эмульсии должен находиться на каждом рабочем месте, где выполняют эпидуральную анестезию или большие блокады нервов высокими дозами МА.

Role of mitochondrion-dependent apoptosis in reduction of bupivacaine-induced cardiotoxicity by lipid emulsion in rats

Objective To evaluate the role of mitochondrion-dependent apoptosis in reduction of bupivacaine-induced cardiotoxicity by lipid emulsion in rats. Methods Forty-five healthy adult male Sprague-Dawley rats, weighing 300-350 g, were divided into 3 groups by a random number table method: sham operation group (Sham group, n=5), bupivacaine group (B group, n=20), and lipid emulsion group (L group, n=20). Cardiac arrest was induced by intravenously injecting 0.4% bupivacaine 30 mg/kg over 20 s to establish the cardiotoxicity model.Twenty percent lipid emulsion was intravenously injected in a dose of 5 ml/kg during resuscitation in group L, and normal saline was intravenously injected in a loading dose of 5 ml/kg during resuscitation in group B, followed by a 3-min infusion of 1 ml·kg-1·min-1in two groups.The successful resuscitation and survival rate at 120 min of return of spontaneous circulation (ROSC) were recorded.Systolic blood pressure, heart rate, mean arterial pressure, rate-pressure product (RPP) and ratio of RPP at each time point after recovery of spontaneous heart beat to baseline value (RPPh) were recorded every 10 min after ROSC.The time from administration to cardiac arrest (T0), time from beginning of cardiopulmonary resuscitation to appearance of the first spontaneous heart beat (Ts) and time from beginning of cardiopulmonary resuscitation to appearance of ROSC (Tr) were recorded.Rats were sacrificed at 120 min of ROSC, and left ventricular tissues were obtained for determination of the expression of Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, cytochrome C (Cyt c) in cytoplasm and mitochondria (by Western blot) and expression of Bax and Bcl-2 mRNA (by real-time polymerase chain reaction) and for examination of myocardial ultrastructure. Results Compared with Sham group, the expression of Bcl-2 protein and mRNA and mitochondrial Cyt c was significantly down-regulated, and the expression of Bax protein and mRNA, cleaved caspase-9, cleaved caspase-3 and cytoplasmic Cyt c was up-regulated in B group (P 0.05), and the pathological changes of myocardium were significantly attenuated in L group. Conclusion The mechanism by which lipid emulsion reduces bupivacaine-induced cardiotoxicity may be related to inhibiting mitochondrion-dependent apoptosis in rats. Key words: Fat emulsions, intravenous; Bupivacaine; Drug-related side effects and adverse reactions; Mitochondria; Apoptosis

Fatty-acid oxidation and calcium homeostasis are involved in the rescue of bupivacaine-induced cardiotoxicity by lipid emulsion in rats*

Lipid emulsion has been shown to be effective in resuscitating bupivacaine-induced cardiac arrest but its mechanism of action is not clear. Here we investigated whether fatty-acid oxidation is required for rescue of bupivacaine-induced cardiotoxicity by lipid emulsion in rats. We also compared the mitochondrial function and calcium threshold for triggering of mitochondrial permeability transition pore opening in bupivacaine-induced cardiac arrest before and after resuscitation with lipid emulsion. Prospective, randomized animal study. University research laboratory. Adult male Sprague-Dawley rats. Asystole was achieved with a single dose of bupivacaine (10 mg/kg over 20 secs, intravenously) and 20% lipid emulsion infusion (5 mL/kg bolus, and 0.5 mL/kg/min maintenance), and cardiac massage started immediately. The rats in CVT-4325 (CVT) group were pretreated with a single dose of fatty-acid oxidation inhibitor CVT (0.5, 0.25, 0.125, or 0.0625 mg/kg bolus intravenously) 5 mins prior to inducing asystole by bupivacaine overdose. Heart rate, ejection fraction, fractional shortening, the threshold for opening of mitochondrial permeability transition pore, oxygen consumption, and membrane potential were measured. The values are mean ± SEM. Administration of bupivacaine resulted in asystole. Lipid Emulsion infusion improved the cardiac function gradually as the ejection fraction was fully recovered within 5 mins (ejection fraction=64±4% and fractional shortening=36±3%, n=6) and heart rate increased to 239±9 beats/min (71% recovery, n=6) within 10 mins. Lipid emulsion was only able to rescue rats pretreated with low dose of CVT (0.0625 mg/kg; heart rate~181±11 beats/min at 10 mins, recovery of 56%; ejection fraction=50±1%; fractional shortening=26±0.6% at 5 mins, n=3), but was unable to resuscitate rats pretreated with higher doses of CVT (0.5, 0.25, or 0.125 mg/kg). The calcium-retention capacity in response to Ca²⁺ overload was significantly higher in cardiac mitochondria isolated from rats resuscitated with 20% lipid emulsion compared to the group that did not receive Lipid Emulsion after bupivacaine overdose (330±42 nmol/mg vs. 180±8.2 nmol/mg of mitochondrial protein, p<.05, n=3 in each group). The mitochondrial oxidative rate and membrane potential were similar in the bupivacaine group before and after resuscitation with lipid emulsion infusion. Fatty-acid oxidation is required for successful rescue of bupivacaine-induced cardiotoxicity by lipid emulsion. This rescue action is associated with inhibition of mitochondrial permeability transition pore opening.

Betulinic Acid has an Inhibitory Effect on Pancreatic Lipase and Induces Adipocyte Lipolysis

Betulinic acid is a pentacyclic triterpenic acid that exists naturally in many kinds of food and has many biological functions. The present study investigated the antiobesity properties of betulinic acid and possible mechanisms by which betulinic acid functions. To examine the antilipase function of betulinic acid, the ability of betulinic acid to inhibit pancreatic lipase activity in vitro and to prevent the elevation of plasma triacylglycerol levels was tested after oral administration of a lipid emulsion in rats. In addition, the lipolytic effects of betulinic acid were assayed in rat adipose tissues. The activity of cAMP-dependent phosphodiesterase was also measured in vitro. Betulinic acid inhibited pancreatic lipase activity in a dose-dependent manner at concentrations of 1.5-100 µM (IC₅₀ value of 21.10 µM) and prevented the elevation of plasma triacylglycerol levels 2 h after oral administration of the lipid emulsion at a dose of 100 mg/kg. In addition, betulinic acid had a strong lipolytic effect, which was mediated by cAMP-dependent phosphodiesterase inhibition. In conclusion, betulinic acid may exert antiobesity effects by directly inhibiting pancreatic lipase, which would prevent the absorption of lipid from the small intestine. In addition, it was found that betulinic acid may further accelerate fat mobilization by enhancing the levels of lipolysis in adipose tissues.

Dosage plan of a flurbiprofen injection product using inhibition of protein binding by lipid emulsion in rats

Flurbiprofen-axetil (FP-ax), a bolus injection product of a non-steroidal anti-inflammatory drug (NSAID), is a prodrug of flurbiprofen, an NSAID. As flurbiprofen strongly binds to site II of human serum albumin (HSA), the free (unbound) concentration of flurbiprofen after injection of FP-ax is low. We have examined the inhibitory effect of free fatty acid (FFA), a binding inhibitor for site II of HSA, on the binding of flurbiprofen in-vitro and in-vivo by ultrafiltration, to establish an effective dosage of FP-ax. In-vitro, fatty acid mixtures (FAs) inhibited the binding of flurbiprofen to rat serum albumin. The free fraction of flurbiprofen was remarkably increased by FAs in rat serum. In-vivo, FP-ax was injected into a control group (low FFA concentration in serum) and a lipid emulsion group (high FFA concentration in serum). The area under the curve of the free concentration of flurbiprofen during the alpha phase and the distribution volume of the central compartment of flurbiprofen were significantly higher in the lipid emulsion group than the control group (5.0- and 1.2-times, respectively). When FP-ax was administered at high FFA concentration, the free concentration of flurbiprofen and distribution of flurbiprofen to tissues increased transiently. This administration method may be useful for patients with cancer pain, having a potent analgesic effect.

Effect of Injection Volume on the Pharmacokinetics of Oil Particles and Incorporated Menatetrenone after Intravenous Injection as O/W Lipid Emulsions in Rats

Oil-in-water lipid emulsions are promising drug carriers for lipophilic drugs, however, the pharmacokinetics after entering the circulation should be clarified at clinical injection volume in order to utilize them in a clinical situation. In the present study, the standard lipid emulsions, consisting of soybean oil, egg yolk phosphatides and menatetrenone with diameters of about 150nm, were prepared using a microfluidizer system. The pharmacokinetics of menatetrenone and the oil particles after intravenous injection as standard lipid emulsions at various injection volumes, from the clinical injection volume (0.1 ml/kg) to the experimental injection volume (3.0 ml/kg), were examined in rats.The plasma concentrations of menatetrenone and the oil particles were similar after administration, showing that menatetrenone was not released even after entering the circulation. Menatetrenone was delivered to the liver and spleen at the clinical injection volume, and more menatetrenone was delivered to the liver at clinical injection volume compared with the experimental volume. Moreover, additional information on injection volume-dependency was also obtained from these findings. These results at various injection volumes suggested that the standard lipid emulsions can be utilized as a useful drug delivery system at the clinical injection volume, especially for liver and spleen targeting.

Sterol side chain length and structure affect the clearance of chylomicron-like lipid emulsions in rats and mice

In previous work we found that sterols such as cholesterol were essential for physiological plasma clearance of lipid emulsions mimicking the structure of mammalian triglyceride-rich lipoproteins. In the present study we compared the clearances of emulsions prepared with sterols of varying alkyl chain length (straight chains, n–C3 to n–C7, or branched chains, i–C5 to i–C10) at the C-17 position. Our studies show that the length of the alkyl chain at the C-17 position of sterols markedly affects the removal of remnant particles from the plasma of rats traced by emulsion cholesteryl oleate label. An alkyl chain of 7 carbons or more was needed for normal remnant clearance. Straight and branched chains of similar length were cleared similarly, showing that the presence of a branch at the end of the alkyl chain had no effect on remnant clearance. For side chains of 7 carbons or less, substitution of sterols with an unsaturation in the alkyl chain close to the terminal carbon markedly decreased the clearance of remnants. Triolein label was used to estimate lipolysis of the injected emulsions. Lipolysis was little affected by the structure of the sterol side chain, except that lipolysis was markedly higher with emulsions containing sterols with an alkyl chain having 4 carbon atoms (n–C4) or with an unsaturation in the 4 carbon alkyl chain. We conclude that the length of the alkyl side chain is an important element in the essentiality of cholesterol as a regulator of metabolism of lipid emulsion models of triglyceride-rich lipoproteins.—Martins, I. J., C. Vilchèze, B-C. Mortimer, R. Bittman, and T. G. Redgrave. Sterol side chain length and structure affect the clearance of chylomicron-like lipid emulsions in rats and mice.

Long-Term Adaptation to High-Fat Diets Modifies the Nature and Output of Postprandial Intestinal Lymph Fatty Acid in Rats

These studies were designed to investigate the lymph absorption of a lipid emulsion in rats prefed different long-term high-fat diets. Particular emphasis was placed on the consequences of endogenous fatty acid alteration on the lymph recovery of two labeled fatty acids. Male Wistar rats were fed a standard diet (LF) containing 3.5 g/100 g fat or high-fat diets containing 15 g/100 g sunflower oil (HSFO), menhaden oil (HMO) or medium-chain triglyceride oil (HMCT) for 4 wk. The lymph was collected for 3 h before and after the intraduodenal infusion of a 90 μmol lipid emulsion (30 μmol monopalmitin, 30 μmol oleic acid, 25 μmol linoleic acid, 5 μmol arachidonic acid) labeled with [3H] oleic (OA) and [14C] arachidonic (AA) acids. The [3H] OA and [14C] AA lymph recoveries were measured and the lymph samples were tested for fatty acid, phospholipid and triglyceride content. Prefeeding an HSFO or HMO diet led to a 65 or 32% greater total lymph fatty acid output, respectively, compared with rats prefed the LF diet. In rats prefed both the HSFO and HMO diets, lymph fatty acid characteristics provided evidence of a dilution of exogenous fatty acids coming from the emulsion by endogenous fatty acids. In rats prefed the HMCT diet, the total lymph fatty acid output after the infusion of the lipid emulsion was not greater than that of starved rats. Nevertheless, 27% [3H] OA and 21% [14C] AA were recovered in the lymph, suggesting a limited dilution of exogenous fatty acids by endogenous fatty acids. In rats prefed the HMCT diet, some exogenous long-chain fatty acids must have been transported by the portal vein in response to low biliary phopholipid production, as indicated by the proportions of [3H] OA and [14C] AA taken up by the mucosa and not recovered in the lymph. Thus we demonstrated that during absorption of a single long-chain fatty acid meal a dilution of exogenous fatty acids by endogenous fatty acids occurred. The nature and the quantity of these endogenous fatty acids could alter the absorption efficiency of long-chain fatty acids by the lymphatic pathway and modify the fatty acid characteristics of lymph lipoprotein.

Pharmacokinetics of Highly Lipophilic Antitumor Agent Palmitoyl Rhizoxin Incorporated in Lipid Emulsions in Rats.

The effects of i.v. formulations on the pharmacokinetics were examined for two antitumor agents with different lipophilicities: rhizoxin and palmitoyl-rhizoxin (RS-1541). Blood disposition and tissue distributions in rats were evaluated using three formulations: polyethylene glycol 400 (PEG)/dimethylacetamide (DMA) solution, colloidal solution, and lipid emulsions composed of dioctanoyl decanoyl glycerol (ODO) and polyoxyethylene-(60)-hydrogenated castor oil (HCO-60). The effects of emulsion particle size on the pharmacokinetics were also investigated. Rhizoxin rapidly disappeared from the plasma and showed high distribution in the tissues, and in vitro rapidly degraded in the plasma independent of the formulations used. In in vitro plasma, rhizoxin was easily released from the emulsion particles. In contrast to rhizoxin, the pharmacokinetics of RS-1541 with greater lipophilicity changed considerably depending on the formulations. The emulsions showed high and sustained plasma concentrations for RS-1541. RS-1541 was stably incorporated in the emulsion droplets and protected from the degradation when it was applied as an emulsion. Tissue distributions of RS-1541 in rats after an injection as lipid emulsion were strongly affected by the emulsion particle size. Small size emulsions (100-110 nm) showed the highest plasma concentrations of RS-1541, though they were unable to suppress distributions of the drug in peripheral tissues. Emulsions larger than 200 nm (approx.) in size, on the contrary, effectively inhibited the drug from entering the bone marrow, small intestine and other non-reticuloendothelial system (non-RES) organs, where many cytotoxic compounds showed undesired toxicities. These results indicate that the lipid emulsions composed of ODO and HCO-60 could be a promising and effective DDS carrier for RS-1541, which is highly lipophilic and stabilized in the emulsions. This was not the case for rhizoxin, however, which was less lipophilic than palmitoyl analogue RS-1541. The work described herein has demonstrated that by properly selecting the particle size, these lipid emulsions can control the behavior of a drug in the body.

Clearance from plasma of triacylglycerol and cholesteryl ester after intravenous injection of chylomicron-like lipid emulsions in rats and man.

Chylomicrons transport fat and cholesterol via lymphatic vessels from the intestine into the bloodstream. The understanding of the metabolism of chylomicrons in man has been slowed by the difficulty of obtaining lymph chylomicrons for experimental studies. Acceptable methods for the study of chylomicron clearance in man are required, because the metabolism of chylomicrons may be abnormal in diseases such as diabetes mellitus. Metabolism of chylomicrons may also play a role in the development of atherosclerosis. In the present work, lipid emulsions were used as a physical model of chylomicrons. Triacylglycerol-rich lipid emulsions labelled with tracer amounts of radioactive triolein and cholesteryl oleate were prepared by sonication and purified by density gradient ultracentrifugation, then injected into unanaesthetized rats and normal human subjects. Plasma radioactivities were measured for 30 min in rats and 90 min in human subjects. Rat lymph chylomicrons were also injected into rats for comparison with the clearance of the lipid emulsions. The plasma clearance data for triacylglycerols and cholesteryl esters were fitted with a kinetic model using the SAAM/CONSAM programs. Multiple studies analysis of the individual studies in each group was used to obtain estimates of the parameter average values and variabilities. The plasma residence times of the lipid labels were obtained from the fitted clearance data. Our results suggest that information about chylomicron metabolism in man can be obtained by analysis of the plasma clearance data following the injection of suitably labelled chylomicron-like lipid emulsions. Our data provide a baseline for comparisons with individuals having abnormalities of lipid metabolism or risk factors for arteriosclerosis.

Effects of sphingomyelin and phosphatidylcholine acyl chains on the clearance of triacylglycerol-rich lipoproteins from plasma. Studies with lipid emulsions in rats

Series of lipid emulsions were prepared as physical models of lymph chylomicrons. The emulsion phospholipid was systematically varied with respect to sphingomyelin, in 0-100% mixtures with egg yolk phosphatidylcholine (EYPC). In other emulsions, the phospholipid was systematically varied with respect to dipalmitoylphosphatidylcholine (DPPC) in 0-100% mixtures with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). All emulsions contained unlabeled free cholesterol, radiolabeled triolein (TO) and radiolabeled cholesteryl oleate (CO). The emulsions were injected into conscious rats to measure the clearances of emulsion TO and CO and the capture of lipid radioactivity by selected organs. The emulsions containing EYPC or POPC were metabolized similarly to lymph chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of emulsion TO followed by hepatic uptake of the CO in the triglyceride-depleted emulsion remnants. Emulsions stabilized with either 1-oleoyl-2-stearoyl- or 1-stearoyl-2-oleoylphosphatidylcholine (OSPC or SOPC) were metabolized similarly. Increasing amounts of sphingomyelin in EYPC emulsions progressively slowed the removal of TO and CO labels from plasma. With 50% sphingomyelin clearance was very slow, while emulsion clearance was negligible with 100% sphingomyelin. Emulsions containing 20% of DPPC in POPC were metabolized similarly to 100% POPC, but 40% or more of DPPC progressively slowed the removal from plasma of both TO and CO. With 100% DPPC clearance was characterized by a rapid initial removal of about 30% of the injected material, followed by a second phase when removal was negligible, suggesting lack of hydrolysis of triacylglycerols by lipoprotein lipase. Changes in the apolipoproteins associated with the emulsions probably mediated the observed changes in clearance.