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PubMed1990

Isolation and characterization of an unique kidney antigen of relevance in human renal disease.

Recently we described a monoclonal antibody, designated 7-5Q, with specificity for a human non-glomerular basement membrane capillary wall antigen. In order to purify and characterize the corresponding antigen for the development of sensitive ELISA techniques, applicable to the diagnosis and monitoring of renal disease, human kidney cortices were extracted with a variety of detergents. A double band of 98/105 kD was evident by immunoblotting in all preparations most notably with a Triton X-114 extract. The caprylic acid- and ammonium sulfate-purified monoclonal antibody 7-5Q was covalently bound to CNBr-activated Sepharose and detergent extracts were applied to this affinity material. A pure protein of 98/105 kD (double band on SDS-polyacrylamide gels) was eluted. Glycan typing with lectins revealed N-acetyl glucosamine-residues and amino acid analysis a relatively high content of acidic (31%) and hydrophobic (30%) amino acids, indicating that the antigen is an acidic, membrane-bound glycoprotein.

Amino Acids↗
PubMed1990

Stopped-flow ESR study on the reactivity of vitamin E, vitamin C and its lipophilic derivatives towards Fremy's salt in micellar systems.

The reaction between Fremy's salt and alpha-tocopherol (VE), ascorbic acid (VC) and its lipophilic derivatives ascorbyl-6-caprylate (VC-8), 6-laurate (VC-12) and 6-palmitate (VC-16) were studied by stopped-flow ESR spectroscopy in cetyl trimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) micelles, as a model reaction of these antioxidants with alkyl peroxy radicals in biological systems. The second order rate constants for the reaction of Fremy's salt with VE in CTAB and SDS micelles were found to be 7.9 x 10(3) and 2.2 M-1 s-1, respectively, with as high as a 3600-fold variation. Rate constants for VC, VC-8, VC-12 and VC-16 are 4.3, 35, 53 and 56 x 10(3) M-1 s-1 and 3.3, 2.7, 1.2 and 0.86 x 10(3) M-1 s-1 in CTAB and SDS micelles, respectively. The results demonstrate remarkable effects of the charge type of the micelles and the side-chain of the antioxidants on the antioxidation reactivity in the micelles. It reveals that the inter-micellar diffusion may be the rate-limiting step for antioxidation carried out in micelles.

Antioxidants↗
PubMed1990

Non-linear least squares analysis of phase diagrams for non-ideal binary mixtures of phospholipids.

A computer program for non-linear least squares minimization has been applied to construct temperature-composition phase diagrams for several binary systems of different phospholipids based on their calorimetric data. The calculated phase diagram is guided to fit the calorimetric data with two adjustable parameters that describe the non-ideal mixing of lipid components in the gel and liquid-crystalline phases. The parameter estimation procedure is presented to show that the computer program can be used not only to generate phase diagrams with characteristic shapes but also to numerically estimate the lipid-lipid pair interactions between the mixed and the like pairs in the two-dimensional plane of the bilayer in both the gel and liquid-crystalline states. The binary lipid systems examined include dimyristoylphosphatidylcholine/1-palmitoyl-2-stearoylphosphatidylchol ine, 1-capryl-2-behenoylphosphatidylcholine/1-behenoyl-2-lauro ylphosphatidylcholine, and 1-stearoyl-2-caprylphosphatidylcholine/dimyristoylphosphatidylchol ine.

Chemical Phenomena↗
PubMed1987

The effect of fatty acid chain length on the rate of arylester hydrolysis by various albumins.

A series of synthetic chromogenic and fluorogenic substrates for monitoring the arylesterase-like activity of albumins from various sources has been studied. Except for ovalbumin, all displayed enzyme-like activity. The acetate, butyrate, caprylate, laurate, and palmitate esters of a coumarin dye were found to be efficiently hydrolyzed within the pH range 8.8-9.8, with the non-enzymatic rate being highest for acetate and lowest for laurate. The latter is considered to be the substrate of choice because it is cleaved most quickly by the proteins tested. A considerable increase in hydrolytic activity was observed upon addition of detergents, but not of Ca and Mg ions, while addition of 1 equivalent of lauric acid to BSA resulted in a 30% decrease in its activity. The results are interpreted in terms of the well-known affinity of albumins for long chain fatty acids and provide the basis for a sensitive determination of small amounts of albumins.

Animals↗
PubMed1983

Coenzyme A in dog kidneys during hypothermic perfusion.

In kidneys perfused hypothermically for 48 hr, both major intracellular pools of CoA (present in cytosol and mitochondria) decreased when solutions with intracellular or extracellular electrolyte composition without CoA precursors were used. The presence of substrates such as glucose or caprylic acid in the perfusate did not counteract the depletion of CoA. The addition of the CoA precursors pantethine and adenosine together with respiratory substrates to the perfusion medium counteracted the loss of CoA. The best solution in this respect among 16 different perfusates tested contained palmitic acid and L-carnitine together with CoA precursors and dibutyryl cyclic AMP. With this solution, we obtained good preservation of the cytosolic and the mitochondrial pool of CoA.

Animals↗
PubMed1988

Synthesis of acylated SOD derivatives which bind to the biomembrane lipid surface and dismutate extracellular superoxide radicals.

Involvement of oxygen radicals in the pathogenesis of various inflammatory diseases has been the focus of recent attention. Since lipid peroxidation of cell membranes is postulated to be one of the major reasons for radical-induced tissue injury, inhibition of oxygen toxicity at or near plasma membranes is important. To metabolize extracellular superoxide radicals effectively at or near cell membranes, we synthesized amphipathic superoxide dismutase (SOD) derivatives (AC-SOD) by covalently linking hydrophobic fatty acids with different chain lengths, such as caprylic acid, capric acid, lauric acid and myristic acid, to the lysyl amino groups of the enzyme. When incubated with erythrocytes or polymorphonuclear leukocytes (PMNs), AC-SOD, but not SOD, bound to plasma membranes of these cells. When topically instilled to the eye, AC-SOD also bound to corneal epithelial cell surface. Upon activation by phorbolmyristyl acetate, extracellular cytochrome c was rapidly reduced by PMNs which were pretreated with SOD. In contrast, PMNs preincubated with AC-SOD failed to catalyze the reduction of cytochrome c under the same experimental conditions. These results suggested that AC-SOD bound to cell membranes and effectively dismutated superoxide radicals at or on the outer surface of plasma membranes.

Acylation↗
PubMed1991

Intestinal effects of the products of lipid digestion on gastric electrical activity in the cat. Possible involvement of vagal intestinal receptors sensitive to lipids.

The relationship between the lipid content of the intestinal lumen and gastric motor activity was studied in anesthetized cats. For this purpose the electromyographic activity was recorded in the antrum whereas the small intestine (duodenum and first part of jejunum or ileum) was perfused with various solutions including calcium propionate, tributyrin, sodium caprylate, potassium oleate, mixtures containing linoleic acid, monolein, triolein, mixture of triglycerides, and glycerol. Long-chain lipids and glycerol both induced a decrease in the rate of basal antral activity. This effect was found to depend on the state of lipid hydrolysis (triglycerides, monoglycerides, and fatty acids) and was most marked with monolein, linoleic acid, and glycerol. With the middle-chain lipids used, these effects were only slight; short-chain lipids did not induce any visible changes in gastric activity. All these effects were prevented by cervical bivagotomy. It was concluded that the two types of lipid-sensitive vagal receptors, which we recently showed to exist in the small intestine, trigger an inhibitory enterogastric reflex that contributes to the regulation of gastric emptying.

Animals↗
PubMed1991

Production and isolation of guinea pig IgE antibody.

Immunoglobulins of the IgE and IgG classes have been causally associated with hypersensitivity reactions in man and in numerous animal species including mice, rats and guinea pigs. The use of the guinea pig as an animal model for both pulmonary and dermal hypersensitivity reactions, and the recent recognition of the importance of IgE antibodies in both early- and late-onset hypersensitivity responses, has heightened interest in production, separation, and isolation of this immunoglobulin class from the guinea pig. IgE antibodies were produced by treatment of strain 13 guinea pigs with cyclophosphamide followed by injection with S. aureus enterotoxin. Serum was obtained and the globulin fraction isolated by addition of caprylic acid then ammonium sulfate. Immunoglobulins were separated into classes using fast protein liquid chromatography (FPLC) employing a Mono Q column and a linear gradient of 0.01-0.3 M Na,K phosphate buffer, pH 7.5 (buffer B). IgG eluted in two major peaks. IgG2 was not retained on the column and emerged with the starting buffer; IgG1 was eluted with 15-20% buffer B. IgE, detected as heat labile homocytotropic antibody, was found in the fraction eluting with 30-35% buffer B. The elution profile of the guinea pig immunoglobulins was predicted from the pattern obtained with immunoglobulin classes from other species. This chromatographic procedure enabled rapid isolation of immunoglobulin classes from guinea pig sera and effectively separated IgG1 from IgE, the two classes associated with hypersensitivity reactions.

Animals↗
PubMed1995

Prospective evaluation of the absorptive capacity of the bowel after major and minor resections in the neonate.

The outcome after major bowel resection in the neonatal period depends primarily on the time needed for bowel adaptation. A prospective study was begun in neonates after small bowel resection to evaluate the absorptive capacity of the bowel and growth parameters as a result of adaptation of the bowel. Twenty-four neonates who underwent bowel resection were included. The underlying diagnoses were necrotising enterocolitis (12), jejunal atresia (3), meconium peritonitis (3), and other (6). During the study, a standardized treatment with respect to nutrition was followed. At predetermined times, enterostomy fluid or faeces were collected for analysis of carbohydrate content, fat content, and fatty acids, together with a xylose test and a hydrogen breath test. Growth parameters included weight and height. The 24 patients were divided into two groups. Group A consisted of eight patients with short bowel syndrome (defined as loss of more than 50% of the original small bowel length related to gestational age). The mean remaining small bowel length in this group was 34.0% (24% to 42%). Group B consisted of 16 patients who had only minor bowel resections. Retarded growth was observed in four group A patients. Low levels of carbohydrate absorption were found 2 and 4 weeks after the initial operation in group A, and 2 weeks after the initial operation in group B. Low levels of fat absorption were found 4 and 8 weeks after the initial operation in group A. The coefficient of absorption of the different fatty acids showed complete absorption of caprylic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Case-Control Studies↗
PubMed1992

Insulinotropic potency of lauric acid: a metabolic rationale for medium chain fatty acids (MCF) in TPN formulation.

The need for a better lipid system to satisfy the fuel requirements of patients while avoiding the adverse effects of current systems has led to suggestions that medium chain fatty acids (MCFs) be incorporated into TPN-lipid emulsions. Since clinical situations requiring TPN are associated with metabolic processes mediated by insulin, in the present study we have therefore examined the effects of a variety of medium chain fatty acids on insulin release. Using an isolated perifused mouse islet model, various doses of medium chain fatty acids and the essential fatty acid, linoleic acid, were tested and compared. The possibility of an additive effect of an insulinotropic MCF and linoleate when both are provided together was also examined. Effluent perifusate samples collected on ice during these experiments were assayed for insulin by radioimmunoassay. It was found that the ability of 5 mM of a given MCF to stimulate insulin secretion was dependent upon its chain length. Thus, while adipic acid (C6) had no effect, Caprylic acid (C8) had a minimal effect that was not statistically significant, but capric acid (C10) and lauric acid had very potent effects that were of the same magnitude to the effect of linoleate on insulin secretion. When insulin output was assessed as the mean integrated area under the curve during a 20-min perifusion, 5 mM lauric acid enhanced insulin secretion from a basal 7351 +/- 666 pg to 15,756 +/- 1680 pg (P less than 0.01, n = 5). In the same experiments, 5 mM linoleic acid stimulated insulin release to 11,260 +/- 867 pg (P less than 0.05). When C12 and linoleate were added together, each at a submaximally effective concentration of 2.5 mM, insulin output was 12,712 +/- 1011 pg (P less than 0.05, n = 5), which was not statistically different from the values obtained when the islets were perifused with 5 mM of each fatty acid alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab')2 fragments.

IgG and F(ab')2 fragments were prepared from horse plasma rich in specific antibodies against Brazilian Bothrops or Crotalus venoms. Both preparations, free of gross contamination with non-immunoglobulin proteins, were able to combine in vitro with their respective antigens, forming immune complexes at antigen excess, equivalence or antibody excess, and activating the C system, through either the classical or the alternative pathways. The IgG preparation was more effective in neutralizing the lethal factors in Bothrops or Crotalus venoms, compared with the F(ab')2 fragments. In contrast, IgG and F(ab')2 anti-Bothrops venom were almost equipotent in neutralizing the haemorrhagic and defibrinating activities in the venom. The method used to purify IgG, precipitation of most non-immunoglobulin plasma proteins with caprylic acid, produced antivenoms richer in specific antibodies, with higher specific activity, recovery and yield, compared with the method commonly used to prepare antivenoms containing F(ab')2.

Animals↗
PubMed1986

Porcine IgG. Isolation of two IgG-subclasses and anti-IgG class- and subclass-specific antibodies.

To investigate the existence of two or more porcine IgG (PIgG) subclasses PIgG was isolated from whey and serum by precipitation with caprylic acid followed by ion-exchange chromatography. Fractions were purified by means of affinity chromatography resulting in the isolation of two products with partly different antigenic determinants. Double immunodiffusion (DI) experiments showed that these products belong to the IgG isotype. Using these products in DI together with antibodies against PIgG class- and subclass-specific determinants it was proven that at least two PIgG subclasses exist (tentatively called IgG1 and IgG2). Quantitation of IgG2 in porcine whey and serum by radial immunodiffusion yielded IgG2 values of 67 and 14 mg/ml, respectively. Probably due to the existence of PIgG1-subpopulations this subclass could not be quantitated reliably. Quantitation of IgG using IgG class-specific antibodies yielded higher IgG values for porcine whey (89 mg/ml) and serum (32 mg/ml) than generally cited in the literature. The possible explanations for these discrepancies are discussed.

Animals↗
PubMed1988

Enzymatic characterization of three aeromonas species using API Peptidase, API "Osidase," and API Esterase test kits.

An enzymatic characterization of 16 strains of Aeromonas species including A. hydrophila (7), A. sobria (5), and A. caviae (4) was carried out using API Peptidase (strips numbered 1, 2, 3, 4, 5, and 6); API Esterase and API "Osidase" test strips. A total of 89 substrates was used in the assay and included 59 arylamides (aminopeptides), 10 esters, and 20 carbohydrates. All three species were remarkably uniform in their reactivities. Nineteen (32%) of the arylamide substrates used were hydrolyzed by all three species. Very strong arylamidase activity was displayed by all three species for L-lysine, L-hydroxyproline, L-arginine, L-alanine, L-proline, and L-leucyl-L-alanine. Esterase activity was strongest against caproate (C6), caprylate (C8), nonanoate (C9), and caprate (C10) substrates. Only a limited number of carbohydrate substrates were hydrolyzed; strong N-acetyl-beta-D-glucosaminidase activity was given by all strains. Both A. hydrophila and A. caviae gave strong beta-D-glucosidase reactivities, while A. sobria appeared to be negative for this enzyme. The results of our preliminary study show that some of the enzymes examined may be useful in the identification and differentiation of these species. The API enzyme assays yielded rapid (4 hr) results. The assays were easy to perform, relatively inexpensive and reproducible. The importance of replicate testing and the inclusion of uninoculated (buffer only) controls as part of the assay is emphasized.

Aeromonas↗
PubMed1994

Influence of the phospholipid n-6/n-3 polyunsaturated fatty acid ratio on the mitochondrial oxidative metabolism before and after myocardial ischemia.

The influence of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on heart pump function and mitochondrial energy metabolism was investigated before and after ischemia. Weanling male Wistar rats were fed for 8 weeks a diet containing either 10% of sunflower seed oil (SSO group) or 10% of a 1:1 (w/w) mixture of fish oil and sunflower seed oil (FO group). The hearts were perfused according to the working mode for 15 min with a Krebs-Henseleit medium containing glucose (11 mM), insulin (10 IU/L) and caprylic acid (25 microM). They were then either maintained in normoxic conditions (70 min) or subjected to a global no-flow normothermic ischemia (20 min) followed by reperfusion (50 min). The aortic and coronary flows were monitored at 5-min intervals. The lactate dehydrogenase (LDH) release in the coronary effluent was evaluated in the control hearts and during ischemia/reperfusion. At the end of the perfusion, two subpopulations of mitochondria were prepared from each heart, by either mechanical or enzyme extraction (ME and EE mitochondria, respectively). The succinate dehydrogenase (SDH) activity was evaluated. Furthermore, the respiration parameters were assessed with either glutamate (20 mM) or palmitoylcarnitine (25 microM) as substrate. Substituting sunflower seed oil by fish oil in the diet provoked a large decrease in the n-6/n-3 PUFA ratio of cardiac phospholipids. The n-3 PUFA enrichment did not alter the coronary and aortic flows nor the LDH release in physiological conditions. Conversely, during post-ischemic reperfusion, the increased amount of n-3 PUFA improved the recovery of aortic flow and decreased the LDH release, without affecting significantly the coronary flow. In ME and EE mitochondria, the phospholipid n-6/n-3 PUFA ratio was similarly modified by the dietary manipulations. The analysis of total cardiac SDH activity suggested an ischemia-induced oedema, of similar magnitude in the two dietary groups. However, neither dietary manipulations nor ischemia influenced the mitochondrial extraction. Similarly, the parameters of glutamate oxidation were also unaffected. Conversely, with palmitoylcarnitine, post-ischemic reperfusion induced a decrease in both state III respiration rate and energy production which were more important in the EE mitochondria of the SSO group. These results suggest that the recovery of mitochondrial energy metabolism and myocardial pump function during reperfusion may be improved in n-3 PUFA-rich hearts. This could be related to a lower injury in n-3 PUFA-rich membranes. Since cardiac function in physiological conditions was not affected by the diet, fish oil could be considered as a beneficial factor to limit heart injury during ischemia and reperfusion.

Animals↗
PubMed2005

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle.

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O(2) consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.

Animals↗
PubMed2006

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression.

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.

Amino Acid Sequence↗
PubMed2005

Absorption enhancement, mechanistic and toxicity studies of medium chain fatty acids, cyclodextrins and bile salts as peroral absorption enhancers.

The objective of the present investigation was to evaluate an oral 'drug delivery' approach, which involves co-administration of absorption enhancers (AEs). The representative low permeable hydrophilic (biopharmaceutic classification system (BCS) Class III) drugs used in the study comprised of cefotaxime sodium and ceftazidime pentahydrate, whereas low permeable lipophilic (BCS Class IV) drugs include cyclosporin A and lovastatin. AEs from three different chemical classes, namely, medium chain fatty acids (sodium caprylate and caprate), cyclodextrins (beta-cyclodextrin, hydroxypropyl beta-cyclodextrin) and bile salts (sodium cholate and deoxycholate) were evaluated for absorption enhancement efficacy, mechanism of action and toxicity using in vitro everted intestinal sac model. These AEs were found to enhance intestinal permeability of drugs from 2- to 27-fold. Light microscopy studies of intestinal sac incubated with AEs for 120 min revealed morphological changes in absorptive mucosa and rank order of toxicity were cyclodextrins>bile salts congruent with medium chain fatty acids. Fluorescence polarization studies indicated that brush bordered membrane vesicles labeled with lipophilic (DPH, 12AS) and hydrophilic dyes (ANS), when treated with AEs exhibited concentration and time dependent decrease in fluorescence polarization. Total protein released in presence of AEs was more than control but considerably less than EDTA (0.58% w/v), which is known to cause toxic release of proteins from cell. Overall, AEs were found to significantly enhance drug permeability by decreasing lipid membrane fluidity and/or interacting with hydrophilic domains of membrane, and has the potential to improve oral delivery.

Animals↗
PubMed2006

Safety evaluation of a medium- and long-chain triacylglycerol oil produced from medium-chain triacylglycerols and edible vegetable oil.

To reduce the incorporation of dietary lipids into adipose tissue, modified fats and oils have been developed, such as medium-chain triacylglycerols (MCT). Typical dietary lipids from vegetable oils, termed long-chain triacylglycerols (LCT), are degraded by salivary, intestinal and pancreatic lipases into two fatty acids and a monoacyl glycerol; whereas, MCT are degraded by the same enzymes into three fatty acids and the simple glycerol backbone. Medium-chain fatty acids (MCFA) are readily absorbed from the small intestine directly into the bloodstream and transported to the liver for hepatic metabolism, while long-chain fatty acids (LCFA) are incorporated into chylomicrons and enter the lymphatic system. MCFA are readily broken down to carbon dioxide and two-carbon fragments, while LCFA are re-esterified to triacylglycerols and either metabolized for energy or stored in adipose tissue. Therefore, consumption of MCT decreases the incorporation of fatty acids into adipose tissue. However, MCT have technological disadvantages precluding their use in many food applications. A possible resolution is the manufacture and use of a triacylglycerol containing both LCT and MCT, termed medium- and long-chain triacylglycerol (MLCT). This manuscript describes studies performed for the safety evaluation of a MLCT oil enzymatically produced from MCT and edible vegetable oil (containing LCT), by a transesterification process. The approximate fatty acid composition of this MLCT consists of caprylic acid (9.7%), capric acid (3.3%), palmitic acid (3.8%), stearic acid (1.7%), oleic acid (51.2%), linoleic acid (18.4%), linolenic acid (9.0%), and other fatty acids (2.9%). The approximate percentages of long (L) and medium (M) fatty acids in the triacylglyerols are as follows: L, L, L (55.1%), L, L, M (35.2%), L, M, M (9.1%), and M, M, M (0.6%). The studies included: (1) acute study in rats (LD50>5000 mg/kg); (2) 6 week repeat-dose safety study via dietary administration to rats (NOAEL of 3500 mg/kg/day), (3) in vitro genotoxicity studies using Salmonella typhimurium and Escherichia coli (negative at 5000 mg/plate), and (4) a four-week, placebo-controlled, double blind, human clinical trial utilizing 20 test subjects (no effects at 42 g MLCT/day). These data are corroborated by other studies published in the peer-reviewed literature on analogous MLCTs.

Administration, Oral↗