A mathematical model of fatty acid metabolism and VLDL assembly in human liver.

The lipid composition of very-low-density lipoprotein (VLDL) in plasma is crucial for human health. A pre-requisite for the alteration of VLDL composition is a co-ordinated understanding of the complex interactions in VLDL assembly. In order to determine the potential effects of changes in substrate availability on VLDL lipid composition, we constructed, parameterized and evaluated a mechanistic mathematical model of the biosynthesis of triglycerides, phospholipids, and cholesterol esters and the assembly of VLDL in human hepatocytes. Using published data on human liver metabolism, the model was also used to provide insight into the complex process of lipid metabolism and to estimate the affinities of different liver enzymes for different fatty acids (FA). For example, we found that Delta6-desaturase is 19 times more selective for C18:3n-3 than C18:2n-6, stearoyl-CoA-desaturase is 2.7 times more selective for C18:0 than C16:0, Delta5-desaturase desaturates C20:4n-3 preferentially over C20:3n-6 and FA elongase preferentially elongates C18:3n-6. The model was also used to predict the plasma free fatty acid (FFA) composition required to generate a prescribed change in plasma lipoprotein FA composition. Furthermore, the model was tested against a published human feeding trial that investigated the effect of changes in dietary FA composition on human plasma lipid FA composition. The model is a useful tool for predicting the effect of changes in plasma FFA composition on plasma lipoprotein lipid FA composition.

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.

Studies on aromatic amino acid oxidase activity in ram spermatozoa: role of pyruvate as an antioxidant.

Chemical antioxidants and catalase have been shown to be ineffective in improving the motility of ram spermatozoa in a chemically-defined diluent (RSD-1). In an attempt to identify the biochemical basis of this observation, the activity of aromatic amino acid oxidase (AAAO), the enzyme responsible for generation of hydrogen peroxide in ram and bull spermatozoa, has been investigated. Ram spermatozoa contained higher levels of AAAO activity than bull spermatozoa, although the physico-chemical properties of the enzyme were generally similar in both species. Components of the medium had a marked effect upon AAAO activity. In the presence of glutamate and 3-(N-morpholino)propanesulfonic acid (MOPS), AAAO activity was decreased. Pyruvate appeared to increase AAAO activity. This was due to the ability of this substance to destroy hydrogen peroxide. Pyruvate in RSD-1 works as an effective antioxidant and therefore eliminates the need for other antioxidants in the semen diluent.

Motility of ram spermatozoa during storage in a chemically-defined diluent containing antioxidants.

The effects of five antioxidants--Vitamin E (VE), butylated hydroxyanisole (BHA), n-propyl gallate (n-PG), deferoxamine mesylate (Desferal) and catalase (EC 1 . 11 . 1 . 6)--on the maintenance of motility of ram spermatozoa in a chemically-defined ram semen diluent (RSD-1) have been evaluated. VE, n-PG and Desferal inhibited spermatozoal motility. The relative inhibition (i.e., ratio of change in % motility over 24 h between the treatment group and the corresponding control) at equimolar concentrations (100 microM) of Desferal, VE and n-PG were 1.6, 1.8 and 3.6 respectively. BHA had no effect at 10 microM but at lower concentrations, gave a slight improvement in motility in freshly diluted spermatozoal samples and in those stored for 1 day at 15 degrees C. The addition of catalase to RSD-1 was also ineffective in improving the motility of spermatozoa. The lack of beneficial effects of the tested antioxidants suggests that RSD-1 itself may destroy reactive oxygen species and the antioxidant activity of RSD-1 components requires further study.

Effect of antifreeze proteins on the motility of ram spermatozoa.

The effect of the antifreeze peptide type I (AFP) from Winter flounder, Pseudopleuronectes americanus, and the antifreeze glycoprotein (AFGP) from Antarctic cod, Dissostichus mawsoni, was assessed on the motility of ram spermatozoa (Dorset and Dorset x Romney) after chilling (5 degrees C) and after freeze-thawing. During chilling, spermatozoal motility decreased significantly at an AFP or AFGP concentration of 0.1 microgram/ml and at concentrations above 10 micrograms/ml (P < 0.05). Thus, at 5 degrees C these antifreeze proteins can have a mildly cytotoxic effect at certain concentrations. Addition of AFP or AFGP to the freezing medium at concentrations of 0.1 to 10 micrograms/ml significantly reduced the loss in spermatozoal motility that occurs due to the freeze-thaw process (P < 0.001). The effect was not concentration dependent nor did it depend on which antifreeze protein was added. However, due to the cytotoxicity during the chilling stage, only AFP at a concentration of 10 micrograms/ml increased the percentage of motile spermatozoa significantly following freezing and thawing over that of the control (P < 0.05). Mechanisms to explain the effect of these proteins on spermatozoal motility after chilling and after freeze-thawing are proposed.

Interaction of sporidesmin, a mycotoxin from Pithomyces chartarum, with lipid bilayers.

Sporidesmin, a mycotoxin from Pithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A2 (PLA2). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations < 10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (approximately 30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA2 kinetics are consistent with their ability to perturb bilayer organisation.

Preparation of representative homogenates of biological tissues: effect of salt on protein extraction.

A technique for the preparation of representative homogenates of tissues is described. For most soft tissues and biological fluids (liver, kidney, semen) more than 80% of protein was recovered after homogenization in isotonic buffer. With tissues that are harder to homogenize, such as heart, spleen, and skeletal muscle, however, only approximately 40-50% of protein was extracted in this medium. Inclusion of salt or salt plus detergent during the homogenization increased recovery of the protein to levels close to those recorded with soft tissues. The increase represented a true recovery and was not an artifact induced by salt/detergent. This situation is parallel to previously reported results for lipid-rich biological tissues.

Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient.

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.

Protein estimation in tissues containing high levels of lipid: modifications to Lowry's method of protein determination.

A method to estimate protein in detergent-solubilized homogenates of lipid-rich biological samples (e.g., adipose tissue, myelin-enriched fractions of sheep brain) is described. The method is also suitable for samples in which protein is present as a protein-detergent complex. The method involves homogenization of tissue in the presence of a suitable detergent and KCl. Protein is then estimated in an aliquot of this homogenate by Lowry's method in the presence of excess sodium dodecyl sulfate, the solutions being clarified by extraction with ethyl acetate. Protein solubilization by Triton X-100 from adipose tissue was biphasic, extracting two to three times more protein under optimum conditions [1.7 +/- 0.1% (v/v) Triton X-100 and 0.75 M KCl], compared with homogenization without salt and detergent. Unlike adipose tissue, protein solubilization from myelin-enriched fractions of sheep brain peaked at 1% (v/v) Triton X-100, resulting in the extraction of approximately three times more protein than homogenization in the absence of detergent and salt.

A comparison of lipids from liver and hepatoma subcellular membranes.

Subcellular fractions of nuclei, mitochondria, endoplasmic reticulum, plasma membrane and cytosol were prepared from liver and hepatoma 7288CTC. Marker enzyme activities, biochemical compositions and electron microscopy were used to establish purity. Hepatoma NADH: cytochrome C reductase and 5'-nucleotidase exhibited abnormal subcellular distributions. The lipids from the subcellular fractions were examined in detail. Mitochondria and plasma membranes were characterized by elevated percentages of diphosphatidylglycerol and sphingomyelin, respectively, in both tissues. All hepatoma subcellular fractions contained dramatically elevated levels of sphingomyelin and cholesterol, two components that form preferential strong complexes in vitro. The fatty acid composition of hepatoma sphingomyelin differed markedly from liver and, unlike liver, did not exhibit organelle specific compositions. Some hepatoma lipid classes contained reduced percentages of palmitate while others contained higher levels. Hepatoma phosphatidylcholine and phosphatidylethanolamine from organelles contained lower percentages of long chain polyunsaturated fatty acids than liver. Generally, unique fatty acid profiles exhibited by individual phospholipid classes of liver subcellular fractions were absent or much reduced in the hepatoma. The ratios of oleate to vaccenate were near one for most of the phospholipid classes of most liver fractions, but all hepatoma classes, with few exceptions, contained a much higher percentage of oleate in all subcellular fractions. The hypothesis is proposed that the origin of some acyl moieties for the biosynthesis of various hepatoma lipid classes differs from liver sources. The possible changes in acyl pools, sources and compartments for complex lipid biosynthesis could result in change in the quantities of molecular species that could contribute to the abnormal properties of the hepatoma membranes.

Colorimetric estimation of inorganic phosphate in colored and/or turbid biological samples: assay of phosphohydrolases.

A simple method of inorganic phosphate determination for colored and/or turbid biological samples is described. The procedure is mild, and so is suitable for routine phosphohydrolase assays. Following deproteinization by ice-cold trichloroacetic (or silicotungstic) acid, the sample was treated with acid-washed charcoal to remove interference due to color. The phosphate in the colorless supernatant was assayed either by measuring the phosphomolybdate spectrophotometrically at 310 nm, following its extraction in organic solvents or by a modified Fiske and Subbarow method. The turbidity interference in the latter case was eliminated either by centrifugation, by sodium dodecyl sulfate treatment, or by extraction of reduced phosphomolybdate blue color by cyclohexanone. Though deproteinization by silicotungstic acid eliminated the turbidity problem, its use in conjunction with charcoal treatment was not convenient.

Membrane lipids of hepatic tissue. I. Neutral lipids from subcellular fractions of liver and hepatoma 7288CTC.

Enriched subcellular fractions of nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane, and cytosol were prepared from liver and hepatoma 7288CTC taken from male inbred BUF rats. Purity was established by marker enzyme activities and distribution of DNA, RNA, sialic acids, total phospholipids, and cholesterol. The subcellular fractions of hepatoma differed from those of liver: 5'-Nucleotidase activity was elevated in ER and mitochondria, cytosol RNA was increased, and cholesterol was elevated in all hepatoma subcellular fractions. Neutral lipid classes of hepatoma subcellular fractions differed quantitatively from those of liver: Hepatoma nuclei and mitochondria contained elevated levels of free fatty acids (FFA) and triglycerides (TG). Generally, the fatty acid profiles of FFA, TG, and sterol esters from hepatoma subcellular fractions were more uniform and showed less organelle specificity than did liver. Hepatoma FFA and TG contained lower percentages of palmitate and higher percentages of stearate in all subcellular fractions than did liver. The sterol esters from most hepatoma subcellular fractions compared to those from liver were characterized by much higher levels of long-chain fatty acids of 20 carbon atoms or longer. The oleate-to-vaccenate ratio in FFA of liver subcellular fractions exhibited some specificity, but not that of hepatoma subcellular fractions. The oleate-to-vaccenate ratio in the acyl chains of liver and hepatoma TG did not reveal organelle specificity.

Membrane lipids of hepatic tissue. II. Phospholipids from subcellular fractions of liver and hepatoma 7288CTC.

Phospholipids from homogenate, nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane (PM), and cytosol of liver and hepatoma 7288CTC (from inbred male BUF rats) were analyzed for their concentrations, fatty acid compositions of individual lipid classes, and levels of octadecenoate positional isomers. The phospholipid concentrations of hepatoma mitochondria and ER were less than 60% of liver values. Sphingomyelin concentrations were elevated dramatically in hepatoma nuclei, mitochondria, and PM. Hepatoma nuclei and mitochondria contained only 25% or less the concentrations of phosphatidylethanolamine (PE) as those of liver, whereas ER, PM, and cytosol fractions of hepatoma contained equal or greater concentration of PE than did the corresponding liver fractions. The fatty acid profiles of the individual lipid classes were somewhat characteristic of liver organelles but not of hepatoma. Lipid classes thought to be located preferentially on the outer bilayer of liver PM contained lower percentages of polyunsaturated fatty acids than did hepatoma PM. For hepatoma generally the lipid classes tended to exhibit a more uniform fatty acid profile among organelles, the polyunsaturated fatty acid percentages were decreased, and the octadecenoate percentages were increased. Octadecenoates isolated from individual lipid classes of organelles contained high levels of cis-vaccenate, in addition to oleate, and some class and organelle specificity was observed in liver. In contrast, hepatoma octadecenoates exhibited little class or organelle specificity, and much higher oleate concentrations were found in PM phosphatidylcholine, PM PE, and ER PE in hepatomas than in liver.

Effect of 2-hexadecynoic acid on cultured 7288C hepatoma cells.

The effects of 2-hexadecynoic acid on the growth and lipid metabolism of cultured 7288 (HTC) cells have been evaluated. Growth was inhibited by the acetylenic acid: the LD50 was 35-85 microM as determined by two methods at low and high cell densities. Reduced growth did not result from damaged plasma membranes as determined by alpha-amino isobutyrate leakage. DNA synthesis was unaffected by the acetylenic acid and the effect on RNA and protein synthesis appeared to be secondary to the effects on lipid metabolism. The 2-hexadecynoic acid inhibited lipid metabolism of the HTC cells at least at two levels. Data from both mass studies and radioactive acetate distributions in cellular and media lipids indicated that fatty acid elongation and acylation, especially triglyceride synthesis, were inhibited.

Action of phospholipase A2 on unmodified phosphatidylcholine bilayers: organizational defects are preferred sites of action.

The hydrolytic action of the bee venom phospholipase A2 on phosphatidylcholine bilayers is studied under a variety of conditions that introduce alterations in the packing, such as those induced by sonication, gel to liquid crystalline phase transition, and osmotic shock. Two phases of hydrolysis could be resolved under a wide range of experimental conditions. With the various forms of the bilayers one observes only a partial hydrolysis of the total available substrate during the first phase. However, the fraction of the substrate hydrolyzed in the first phase changes with the form of the available substrate, with the amount of the enzyme added, with the temperature, with the phase transition characteristics of the substrate, and by the sonication of the substrate. The second phase of hydrolysis is generally observed when a certain concentration of the products has been produced during the first phase of hydrolysis. These obervations are interpreted to suggest that the bee venom phospholipase A2 preferentially catalyzes hydrolysis of the substrate available at or near the defects in the organization of the substrate in the bilayers.

Intrinsic differences in the perturbing ability of alkanols in bilayer: action of phospholipase A2 on the alkanol-modified phospholipid bilayer.

The kinetic parameters for the steady-state rate of hydrolysis of egg phosphatidylcholine in multilamellar vesicles by bee venom phospholipase A2 are measured in the presence of 27 alkanols and several organic solvents. In general, small nonpolar solutes like enflurane, tetrahydrofuran, benzene, chloroform and diethylether do not promote the hydrolysis of multilamellar vesicles. The rate of hydrolysis shows a biphasic dependence upon the alkanol concentration for all higher (C5-C9) alcohols examined, i.e., an optimal rate of hydrolysis is observed at a characteristic concentration for each alcohol. The alkanol to lipid mole ratio (D/L ratio) in the bilayer at the peak activating concentration of an alkanol was computed from its bilayer/water partition coefficient. The branched chain alcohols induce peak activation of hydrolysis at lower D/L ratios in the bilayer than the corresponding straight chain analogs. Similarly, the longer chain n-alkanols at peak activating concentration have a lower D/L ratio than the corresponding lower alcohols. Both the Km and Vm for phosphatidylcholine increase as a function of the chain length of the activating alcohol. These kinetic parameters also depend upon the position of the substituents on the activating alcohols. Both the D/L ratio and Vm for an alcohol are found to change with the cross-sectional area of the activating alcohol across its long axis: alcohols with a more asymmetric cross-section exhibit higher Vm and a lower D/L ratio. Such correlations of Vm and D/L ratio with the molecular parameters of the alkanols are interpreted to suggest that the accessibility of the substrate molecule in the bilayer to the phospholipase is modulated by the free space introduced by the alkanols in the bilayer. Effect of tetradecane derivatives and A2C (a membrane fluidizing agent) on the phase transition characteristics of DPPC bilayers, and their susceptibility to phospholipase A2 from bee venom and pig pancreas is also reported. These solutes cause a broadening of the transition profile and reduce the size of the cooperative unit and the enthalpy of transition. These effects depend upon the mole fraction of a solute in the bilayer; however, equal concentrations of these solutes do not induce equal response. Susceptibility of the modified bilayers to phospholipase A2 depends not only upon the structure of the solute but also upon the source of the enzyme. The data show that the activity of the membrane-bound enzyme is modulated to different extents by different solutes, and the bilayer perturbing ability of these solutes may be related to the asymmetry of their cross-sectional area and to the free space introduced by the alkanols in a bilayer.

Production and mode of action of lactocin 27: bacteriocin from a homofermentative Lactobacillus.

Lactobacillus helveticus strain LP27 produced a bacteriocin, lactocin 27, in dialyzable and nondialyzable forms. No evidence was obtained to indicate that lactocin 27 was under the control of extrachromosomal plasmids. Lactocin 27 had a bacteriostatic effect on the indicator, Lactobacillus helveticus strain LS18. It inhibited primarily protein synthesis without affecting deoxyribonucleic acid and ribonucleic acid synthesis or adenosine 5'-triphosphate levels. Treatment of susceptible cells with the lactocin did not cause leakage of ultraviolet-absorbing material, but caused the efflux of potassium ions and the influx of sodium ions. It adsorbed non-specifically to various bacterial species irrespective of their susceptibility to lactocin 27. However, the presence of specific receptors has not been ruled out.

Isolation and characterization of a bacteriocin from a homofermentative Lactobacillus.

Nearly 100 isolates of Lactobacillus were obtained from human and animal sources. Screening tests with the isolates revealed seven possible bacteriocinogenic strains and 26 strains sensitive to one or more of these inhibitory strains. Three homofermentative strains were selected for additional study after it was shown that their inhibitory substances differed in activity spectrum and in susceptibility to inactivation by proteolytic enzymes. One of these, L. helveticus strain LP27, was shown to produce a potent bacteriocin called lactocin 27. The lactocin was isolated from the culture supernatant fluid as a protein-lipopolysaccharide complex. In the presence of sodium dodecyl sulfate the complex was dissociated, and the activity was found to reside in a small glycoprotein (molecular weight 12,400). The amino acid composition of purified lactocin 27 is similar to that of the L. fermenti bacteriocin; neither requires disulfide bonds for activity.