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High-pressure liquid chromatography of fatty acid esters of retinol isomers. Analysis of retinyl esters stored in the eye.

We have synthesized the all-trans, 13-cis, 11-cis, and 9-cis retinyl esters of some or all of the following fatty acids: caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0), stearic (18:0), arachidic (20:0), palmitoleic (16:1), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic (20:4), and docosahexaenoic (22:6). Mixtures of these compounds were analyzed by high-pressure liquid chromatography on an Altex 100 chromatograph. This approach has enabled us to investigate and identify both the isomeric form and fatty acid basis of the retinyl esters present in rabbit and rat ocular tissues. Dark-adapted rabbits had 11-cis and all-trans retinyl esters, whereas light-adapted albino rats had only the all-trans isomer. The fatty acids utilized for esterification were almost wholly palmitic for the rabbit, whereas both stearic and palmitic were present in the rat esters. Rabbits are similar to frogs in that a large proportion (80%) of their ocular retinyl esters occurred in large, fluorescent oil droplets in the pigment epithelium. These are absent in rats.

Adaptation, Physiological↗

[Oxidation of palmityl carnitine in mitochondria of rat heart and liver tissue under conditions of thyroid pathology].

Effects were studied of various doses of thyroid hormones and thyroidectomy on oxidation of palmityl carnitine and caprylate in heart and liver mitochondria as well as on the rate of oxidation of succinate and alpha-ketoglutarate in brain mitochondria. Depending on the dose of the thyroid hormone the rate of fatty acid oxidation varied. The stimulatory doses of thyroxin and thyroidectomy altered (increased or decreased) the rate of electrone transport in the respiratory chain but the respiratory control and the ADP/O ratio were only slightly changed. Toxic doses led to fast oxidation of the both substrates and to uncoupling of the oxidative phosphorylation. Thyroid hormones and thyroidectomy did not affect the succinate oxidation in brain mitochondria; this data suggest insensitivity of the brain tissue to the effect of thyroid hormones.

Animals↗

Mechanisms of absorption enhancement by medium chain fatty acids in intestinal epithelial Caco-2 cell monolayers.

Sodium salts of medium chain fatty acids (MCFAs) enhance the absorption of hydrophilic drugs across the intestinal mucosa, but the mechanism behind the effect is largely unknown. In this study, the dose-dependent effects of the sodium salts of four MCFAs, C6 (caproate), C8 (caprylate), C10 (caprate) and C12 (laurate), on the permeability of the hydrophilic marker molecule [14C]mannitol were studied in monolayers of the human intestinal epithelial cell line, Caco-2, grown on permeable supports. C8, C10 and C12, but not C6, enhanced the permeability of [14C]mannitol in a dose-dependent manner. Comparison of the cellular effects of the MCFAs at concentrations that gave comparable (8.1- to 8.5-fold) absorption enhancement showed that: 1) C8 was active as absorption enhancer only when the tonicity of the medium was increased; 2) absorption enhancement mediated by C10 was related to a redistribution of the cytoskeleton and structural dilatations in the tight junctions; and 3) C12 was without effect on the cytoskeleton and cellular morphology. Studies on C10 under anisotonic conditions showed that deviations from isotonicity enhanced its effect. These results suggest that structurally similar MCFAs display dramatic differences in their mechanism of action. In addition, the effects of osmolality provide an explanation for the previously reported variability in the efficacy of MCFAs as absorption enhancers.

Caco-2 Cells↗

[The effect of ketalar on fatty acid metabolism in the myocardium].

The intraperitoneal administration of ketalar in a dose of 50 mg/kg elevated the myocardial levels of triglycerides, reduced non-esterified fatty acids and enhanced uncontrolled mitochondrial respiration during oxidation of caprylate and palmitoylcarnitine. The efficiency of phosphorylation remained unchanged.

Animals↗

Problems in pre-transfusion tests related to drugs and chemicals.

Ingestion of drugs can cause patients or blood donors to have a positive direct and sometimes indirect antiglobulin test. The most common cause of these positive reactions and immune hemolytic anemia due to drugs is the formation of red cell autoantibodies. These autoantibodies will react with the patient's own red cells and usually most other normal red cells in vitro without the drug being present. The prototype drug causing this type of reaction is alpha methyldopa (Aldomet). Other drugs cause positive antiglobulin tests by three different mechanisms, the drug antibodies reacting with red cells in vitro only in the presence of the drug. The first of these mechanisms causes positive reactions because the drug binds firmly to the red cell membrane, and antibody against the drug will combine with the drug on the membrane leading to IgG-sensitized red cells. The prototype drug for this mechanism is penicillin. The second mechanism involves chemical modification of the red cell membrane by the drug so that it takes up many proteins nonspecifically; the cephalosporins are the only group of drugs known to react in this fashion. The final mechanism involves the formation of an immune complex by the drug and its specific antibody. This immune complex will attach to cell membranes, usually activating complement in the process. Examples of drugs thought to operate by this mechanism are phenacetin, quinine, and quinidine. Some individuals have antibodies present in their serum that will react with chemical added to commercial blood bank reagents. Examples of these are antibodies to dyes added to ABO typing sera, antibodies to sodium caprylate in bovine albumin, and antibodies to chemicals added to red cell diluents, e.g., chloramphenicol, neomycin, and hydrocortisone. If these antibodies are present they can create problems in pretransfusion testing; in particular, they can present anomalies in ABO, Rh grouping, and antibody detection.

Agglutination↗

[Serum fatty acids in premature neonates].

In the past we have published several papers concerning the evolution of large spectrum of fatty acids in the brain or in the blood-serum in rats or in human newborns (and their mothers just after the delivery). Therefore we realized this study: the blood-serum spectrum of fatty acids in the healthy human newborns (n = 32) was compared with the spectrum of fatty acids detected in the blood-serum of human premature neonates (n = 22). The birth-weight in the control group was 3,882, in the premature neonates 2,137 g only. Fatty acids were measured as methylesters (FAME) using the method of gas chromatography and absolutely the same methodological approach as in our previous studies (33, 22, 23). The results were computed by biocybernet. dep. of Physiological institute and the statistics were evaluated by Mann-Whitney U test. It was established that the premature neonates show a distinct greater content (expressed in percentage) of saturated fatty acids and a smaller share of polyenoic FA (PUFA). A further marked difference consists in a significant lower of the n-3 FA and consequently a higher n-6/n-3 index. In premature neonates a significant increase of caprylic acid and arachidonic acid in the blood-serum was detected. The significance of described changes in the participation of single fatty acids in the premature neonates for their maturation, structural evolution especially of the brain cells and for therapy, is discussed.

Fatty Acids↗

Production of extracellular acidic lipase by Rhizopus arrhizus as a function of culture conditions.

Thermophilic strain of Rhizopus arrhizus accumulates an acidic lipase in culture fluid when grown in a medium containing ground nut oil, milk powder and inorganic salts. Addition of 2.0% ground nut oil yielded the highest productivity of enzyme. Soyabean meal and arabinose were found to be the best nitrogen and carbon sources for enzyme production respectively. Addition of metal ions such as MnCl2, SnCl2 and CaCl2 increased the enzyme productivity by 4 fold. The enzyme productivity in the fermenter was much higher (310 U/ml) than in shake-flask (180 U/ml). Crude lipase preparation showed pH and temperature activity optima at 3.5 and 45 degrees C respectively. The enzyme is thermostable and highly active in hydrolysing triglycerides and failed to hydrolyse-methyl esters of caprylate and palmitate.

Culture Media↗

X. Lamb and veal. Comprehensive evaluation of fatty acids in foods.

As a result of critical review of information on lipid composition of foods, representative values have been derived for the lipid and fatty acid contents of lamb and veal. Comprehensive tabulations of fatty acids in 100-gm. portions of raw and cooked retail cuts are given. The mean fatty acid profiles of the lean and adipose tissues which were used for computing the fatty acid values in the food tables are presented. The lean tissue lipid of lamb contains a significant amount of caprylic, arachidic, and arachidonic acids which are not present in the adipose tissue. A comparison of beef and veal shows that veal lipid is a richer source of polyunsaturated fatty acids. The new values presented here for fatty acids in lamb and veal should provide nutritionists and dietitians better means for evaluating diets.

Animals↗

[Rapid detection of Salmonella with MUCAP reagent].

The 4-methylumbelliferyl-caprylate (MUCAP) was applied for the identification of Salmonella, it's specification, sensitity, and availability were reported. 65 strains of standard Salmonella, 48 strains of Salmonella isolated from foods growing on plates of HE, DHL, SS and MaConkey agar have been tested with MUCAP. All of them were identified as MUCAP positive; whereas, among the non-Salmonella tested only Pseudamonas spp., Aeromonas hydrophilia and Plesiomonas shigelloides were shown as positive, but they could be differentiated easily with Salmonella by means of oxidase test. The unspecific reaction of Serratia marcescens could be eliminated by using the plate medium which has been added of 1% sucrose. Both the sensitivity and specificity of the method were up to 97% more, and the operating of the method was very facility and to be done within few minutes.

Hymecromone↗

[Determination of the specificity of microbial esterases in vivo].

Gas chromatography is used to determine the specificity of microbial esterases in vivo. The technique is based on hydrolysis by the cells of esters of fatty acids containing different alcohol groups. The specificity was evaluated by means of the coefficient Kc which changed from 0 to 1 and did not depend on the time of hydrolysis. The esterases of M lysodeicticus were more specific than the esterases of S. marcescens by a factor of 30 during hydrolysis of the equimolar mixture of methyl ester of pelargonic acid and ethyl ester of caprylic acid. This criterium of specificity may be used for rapid identification of bacteria.

Bacteria↗

EDTA-resistant and sensitive phosphotriesterase activities associated with albumin and lipoproteins in rabbit serum.

Phosphotriesterase (PTE) activities in mammalian serum are typically found in the lipoprotein fraction. This PTE requires Ca++ for activity and is consequently inactivated by ethylenediaminetetraacetic acid (EDTA). There is also a little known PTE in mammal serum that is resistant to EDTA inactivation. In this work, the PTE activities for the substrates O-hexyl O-2, 5-dichlorophenyl phosphoramidate (HDCP) and O,O'-diethyl p-nitrophenyl phosphate were purified from rabbit serum by ultracentrifugation, molecular exclusion, and anion exchange chromotography. Rabbit serum produced two PTE activities. One was sensitive and the other was resistant to EDTA inhibition. The EDTA-resistant HDCP hydrolyzing activity and paraoxonase activities of rabbit serum were purified to homogeneity. These activities copurified and were associated to albumin. This EDTA-resistant activity exhibited no stereoselectivity in the hydrolysis of HDCP. The EDTA-sensitive activity was isolated in the lipoprotein fraction and stereoselectively hydrolyzed the S-HDCP over the R-HDCP. Other differences between the EDTA-sensitive paraoxonase and HDCP hydrolyzing activity were discovered in response to p-nitrophenylbutkyrate, 5,5-dithio-bis(2-nitrobenzoic acid), caprylic acid, sodium ions, and ammonium ions. This work demonstrates the existence of two well differentiated PTE activities in rabbit serum. One is sensitive to EDTA, stereoselective, and found in the lipoprotein fraction, and the other is resistant to EDTA inhibition and nonstereospecific.

Animals↗

Effect of selected substances on heat-induced aggregation of albumin, IgG and lysozyme.

Compounds capable of inhibiting protein aggregation may find pharmacological applications in the treatment of a number of diseases called protein condensation diseases [Benedek (1997)], which include cataract, biliary and urinary lithiasis and certain rheumatic diseases. We examined the effect of selected compounds on heat-induced aggregation human serum albumin (HSA), IgG and lysozyme. HSA (0.2% w/v in 0.066 M sodium phosphate pH 5.3 at 22 degrees C), IgG (0.5% w/v in 0.066 M Tris pH 8.0 at 22 degrees C), and L (0.2 % w/v in 0.066 M CAPS pH 11.0 at 22 degrees C) were heated for 30 min at 70 degrees C in the presence or absence of different concentrations of the substance under examination and heat-induced aggregation of 100 microl aliquots was evaluated by measuring the absorbance at 595 nm using an automatic microplate reader. In these conditions, inhibition of aggregation could be due to an anti-denaturant effect or to interferences with the aggregation of denatured molecules, as previously described [Saso, Casini et al. (1998)]. However, this distinction may not be pharmacologically relevant when the target of the therapy is the prevention of abnormal phenomena of protein aggregation. Inorganic salts like NaCl and CaCl2 were active on the three proteins (IgG > HSA > L) but many ligands of HSA such as tryptophan, N-acetyl-tryptophan, caprylic acid, capric acid, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid and bendazac were active on their carrier but not on IgG and L, indicating that the latter proteins are more difficult to protect and that specific anti-denaturant and/or anti-aggregant compounds should be developed.

Albumins↗