Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside: Hepatic synthesis of lipoproteins and apolipoproteins

Hepatic synthesis of lipoproteins and apolipoproteins was investigated in male Wistar rats with severe nephrotic syndrome induced by puromycin aminonucleoside by incubating liver slices with a mixture of¹⁴C-amino acids. Labeled lipoproteins were separated by preparative ultracentrifugation from the incubation medium after the addition of carrier plasma. The incorporation of¹⁴C-amino acids into very low density lipoproteins (VLDL) (1.006 g/ml), low density lipoproteins (LDL) (1.006–1.063 g/ml) and high density lipoproteins (HDL) (1.063–1.210 g/ml) was increased in nephrotic liver 6.1-, 5.7- and 5.0-fold, respectively. The measurement of radioactivity associated to apolipoproteins isolated by SDS-PAGE documented an increased incorporation into apolipoprotein E (apoE) of nephrotic VLDL (33.1% vs 20% of the total radioactivity incorporated into VLDL apoproteins) and a markedly increased incorporation into apolipoprotein A-I (apoA-I) of nephrotic HDL (44.3% vs 16.3% of the total radioactivity incorporated into HDL apoproteins). In nephrotic liver, the total incorporation of amino acids into apolipoproteins (apoVLDL+apoLDL+apoHDL) was increased 12.6 times for apoA-I, 6.4 times for apoB, 5.0 times for apoE, 4.2 times for apoC+apoA-II and 2.5 times for apoA-IV. We suggest that, in nephrotic liver: (a) the synthesis of VLDL, LDL and HDL is increased, and (b) the total synthesis of apoA-I is selectively increased when compared to that of the other apolipoproteins. Preliminary reports of this work were presented at the Annual Meeting of the European Society for the Study of the Liver (Düsseldorf, September 13–15, 1979); at the 5th International Symposium on Atherosclerosis (Houston, November 6–9, 1979) and at the Annual Meeting of the Italian Society for the Study of the Liver (Rome, December 14–15, 1979).     

Comparison of ultracentrifugation and gel filtration for the isolation of bovine lipoproteins

Lipoproteins from the plasma of three nonlactating Holstein cows were isolated using either preparative ultracentrifugation or gel filtration chromatography. Lipoprotein classes obtained by ultracentrifugation were very low density plus chylomicra, <1.006 g/ml; low density, 1.007–1.039 g/ml; high density₁, 1.040–1.063 g/ml; and high density, 1.064–1.22 g/ml. These lipoprotein classes were individually applied to an agarose gel column to determine at what volume they eluted in comparison to lipoproteins that were separated after applying total bovine lipoproteins to the column. Three major peaks corresponding to very low density lipoproteins plus chylomicra, low density, and high density lipoproteins resulted after gel filtration of total lipoproteins. Very low density lipoproteins plus chylomicra, obtained by ultracentrifugation, eluted as a single peak, as did low density and high density lipoproteins. However, high density₁ lipoproteins eluted as two peaks. The first peak eluted at the same volume as low density lipoproteins, and the second peak eluted at a volume similar to that of the ascending slope of the high density lipoprotein peak. Results from disc polyacrylamide gel electrophoresis, immunoelectrophoresis and double immunodiffusion of lipoprotein fractions, and SDS polyacrylamide gel electrophoresis of their apoproteins, similarly indicated that the lipoproteins present in the 1.040–1.063 g/ml density interval are a mixture of low and high density lipoproteins rather than a unique class of lipoproteins.     

Separation of perillyl alcohol from the peel of citrus unshiu by supercritical CO2 and preparative high-performance liquid chromatography

To separate perillyl alcohol (POH), a potential anti-cancer agent, the peel of citrus unshiu was extracted by supercritical CO₂ extraction (SCE) system at 50 ‡C, 200 bar and 6 kg CO₂/hr/kg sample. The extracts were partitioned by acetonitrile/hexane (90/10, vol%). POH was eluted in the acetonitrile phase. An open-tubular chromatography with silica gel (40–63 Μm) was used to purify POH from the acetonitrile phase. Mobile phase was hexane/ethyl acetate (90/10, vol%). To obtain POH in a pure form, finally preparative high-performance liquid chromatography was applied. The collection of POH from citrus unshiu peel was achieved on a laboratory-prepared Chromatographic column (300x3.9 mm) packed with 15 Μm C₁₈ preparative packings. The composition of mobile phase was water/acetonitrile (50/50, vol%). The flow rate of the mobile phase was 1 ml/min and UV wavelength was fixed at 205 nm. It was found that the total yield of POH was 1.6xl0^-3 (wt%) as the dry powder of citrus unshiu peel.     

A cross-species comparison of neutral lipid composition of milk fat of prosimian primates

The fatty acid composition of milk fat is known to be affected by dietary and genetic differences, while the milk triacylglycerol structure is believed to be attuned to the needs of the subsequent lipolysis during gastrointestinal passage. The availability of milk samples from eight species of prosimian primates, whose milk triacylglycerol structure had not been analyzed, offered an opportunity to further assess these ideas. The milk samples were collected by manual expression and the lipids extracted with chloroform/methanol (2∶1, vol/vol). The lipid classes were resolved by thin-layer chromatography, and the neutral lipids subjected to detailed analyses by capillary gas-liquid chromatography of fatty acids and molecular species of triacylglycerols using nonpolar and polarizable liquid phases. The milk samples were found to differ greatly in total fat content (4–73%) and in the composition of the neutral liqid classes and molecular species. The concentration of triacylglycerols ranged from 88–95%, free fatty acids from 0.5–10%, alkyldiacylglycerols from 0.5–5.0%, and diacylglycerols, monoacylglycerols and free and esterified cholesterol made up the remainder. The fatty acid chain length ranged from C₈−C₂₄, with palmitic (16–31%) and oleic (13–40%) acids being the major components in most of the species. In all instances, the molecular association of the fatty acids differed from random distribution by a higher proportion of the monoacid (trioleoyl) and diacid (dipalmitoyloleoyl) glycerols. The phylogenetic influences on neutral milk lipid composition, however, remained unclear, as some of the differences between closely related species were greater than those between more distantly related ones. Triacylglycerol structures are abbreviated by listing their three constituent fatty acids in sequence, e.g., PPP, LaOL.     

Hydrocarbons and alcohols of basking shark and pig liver lipids

Four samples of the unsaponifiables of basking shark liver oil were adsorbed on alumina and eluted to yield Fractions 1–5, inclusive. Analyses by temperature programmed GC and by silica gel chromatography showed hydrocarbons in the first four fractions with squalene increasing to Fraction 3 and the pristane level being highest in Fraction 1. Aside from pristane and squalene, other hydrocarbons occurred at levels of 420–750 mg% in the oils on a weight basis, of which about 60% constituted a series of n-paraffins (relative carbon number range: 15.0–38.0) together with smaller amounts of at least one branched chain saturated group. Unsaturated hydrocarbons eluted mainly after squalene. The oils contained up to 460 mg% sterol and 78–270 mg% alcohols of C₁₀ to C₃₀, the ratio of saturated to unsaturated members being about 1.6. The composition of the unsaponifiable lipids of pig liver was quite different from that of the marine oils. It contained 10.6% sterol in addition to 400 mg% alcohols, the latter consisting of 81.8% saturated components (C₁₂ to C₃₁; ratio of saturated: unsaturated members, 4.4). The hydrocarbons comprised 450–700 mg% of the unsaponifiable mixture and squalene, paraffins and additional unsaturated components occurred at levels of 20.6, 24.4 and 11.9 mg%, respectively. The saturated hydrocarbons were high in normal homologs of relative carbon number range, 15 to 36; pristane could not be detected.     

A simple method for the quantitative isolation and resolution of unesterified fatty acids from fats and oils

The unesterified fatty acids (UFA) present in fats and oils are isolated cleanly and without the formation of detectable artifacts due to saponification. The lipid (≤0.5 g) dissolved in 5 ml of n-hexane is passed over a 300-mg bed of Celite impregnated with saturated, aqueous Na₃PO₄ so that the solution passes through the bed in 3–4 min. After the bed has been washed, the UFA are freed from their salts by pumping of HC1 vapor over the bed. The acids are eluted with CH₂Cl₂ and subsequently separated underivatized by gas liquid chromatography. The C₁₀ through C_18.3 acids are separated in <15 min. Recovery of the C₁₀-C_18∶0 and C_18∶1, C_18∶2 and C_18∶3 acids added to fatty acid-free fats and oils in several concentrations was nearly 100%     

Molecular species of wax esters in jaw fat of atlantic bottlenose dolphin,Tursiops truncatus

The jaw fat of the Atlantic bottlenose dolphin (Tursiops truncatus) contains unusual wax esters which can be separated into short chain (<C₂₄) and long chain (>C₂₄) fractions by thin layer chromatography. The short chain wax esters (28 wt. %) have been characterized as a 72∶24∶4 mixture of isovaleroyl, isobutoryl, and 2-methylbutyrol, esters of C₁₄–C₁₈ n- and iso-alcohols. The intact <C₂₄ esters have been resolved into individual molecular species by gas liquid chromatography on open-tubular polyester columns. The long chain wax esters (12 wt. %) contain C₁₀–C₂₂ n- and iso-acids esterified to the same C₁₄–C₁₈ n- and iso-alcohols. Gas liquid chromatography of the intact, hydrogenated >C₂₄ esters on a short JXR column has characterized them according to carbon number and the number of methyl branches they contain.     

Separation and analysis of phospholipids in different foods with a light-scattering detector

A method for the separation of phospholipids (PL) from total lipids by solid-phase extraction (SPE) with reversephase C₈ cartridges is described. The method was validated with a standard mixture of PL and applied to natural food matrixes, such as egg, chicken meat, salami, and ripened cheese. The recovery of PL ranged between 93 and 99.7% and was evaluated by an organic phosphorus spectrophotometric determination. The egg powder PL fraction obtained by SPE contained about 20% (w/w) nonpolar PL material when 100–150 mg of lipids were loaded onto the cartridge. Higher percentages of nonphospholipid components (30–43%) were obtained when the amount of lipids loaded was below or above the 100–150 mg range. The purified PL fractions were analyzed by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. Good HPLC performance was observed even with low-purity SPE fractions (43% nonphospholipid material).     

Plasma high density lipoprotein subgroup distribution in rats fed diets with varying amounts of sucrose and sunflower oil

The effect of varying the dietary sunflower oil/sucrose (SO/SU) ratio on rat plasma lipid concentration and lipoprotein distribution was studied. Four groups of 10 rats were fed for 4 weeks diets with varying SO/SU ratios. Lipoprotein components were then estimated in whole plasma and after cumulative density ultracentrifugation. Whole plasma triacylglycerol (TG), total cholesterol (TC) and free cholesterol (FC) decreased with increasing SO/SU ratio; the CE/FC ratio increased, because CE remained virtually unaltered. Plasma TG-lowering was due to a decrease in VLDL and LDL-TG. Protein, CE and FC in d=1.063–1.100 g/ml (HDL_2b) and d=1.100–1.125 g/ml (HDL_2a) lipoproteins decreased upon increasing the SO/SU ratio. In contrast, in d=1.125–1.200 g/ml (HDL₃) lipoproteins, there was a concomitant increase in these components. Although increasing the SO/SU ratio effected more protein and CE transportation in HDL₃ and less in HDL₂, the total amount of these components in high density lipoproteins (d=1.063–1.200 g/ml) remained constant. Apo A-I and apo C-III decreased in HDL₂ but increased in HDL₃ upon increasing the SO/SU ratio. Also, HDL₂ apo E, and the apo C-II/apo C-III and small apo B/large apo B ratios in VLDL and LDL were lowered by increasing the SO/SU ratio. The hepatic VLDL-TG output during isolated liver perfusion was lowest in rats fed the diet with the highest SO/SU ratio. In perfusate, like in plasma, the VLDL and LDL apo C-II/apo C-III ratio, as well as the small apo B/large apo B ratio, decreased upon increasing the dietary SO/SU ratio. The results indicate that there can be appreciable diet-dependent variations in plasma HDL subgroup distribution in spite of unchanged total HDL levels.     

α-Helical requirements for free apolipoproteins to generate HDL and to induce cellular lipid efflux

The structural requirement has been studied for apolipoproteins in their free form to interact with cells, to generate high density lipoprotein (HDL), and to cause cellular lipid efflux (J. Biol. Chem. 266, 3080–3086, 1991). It is shown that human apolipoprotein (apo) A-IV and apolipophorin III ofManduca sexta cause cholesterol efflux from cholesterol-loaded mouse peritoneal macrophages and reduce intracellularly accumulated cholesteryl ester as a results of forming HDL-like particles with cellular lipids, as do apoA-I, A-II and E. On the other hand, similar to apoC-III, reduced-and-carboxymethylated human apoA-II had no such effect. Thus, apolipoproteins seem to require at least four amphiphilic helical segments per molecule to express this function.     

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10-through 11,13–18∶2

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I₂ catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18∶2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag⁺-HPLC) and gas chromatography (GC). Ag⁺-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12–18∶2. However, one of the 8,10 isomers (8cis, 10trans-18∶2) coeluted with the 9trans,11cis18∶2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag⁺-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13–18∶2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18∶1 18∶2, and 18∶3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.     

Gas-chromatographic analysis of the aliphatic fractions of brown coal wax

The individual composition of the fractions of wax isolated from brown coal from the Sergeevskoe deposit was studied using gas chromatography. It was found that the aliphatic constituents of wax are C₁₄–C₄₂ n-alkanes with high coefficients of oddness, C₁₄–C₃₀ saturated alcohols, C₁₄–C₃₆ higher fatty acids, and esters. The contribution of unsaturated compounds was found. The structure and formation conditions of coal are considered.     

Four-factor response surface optimization of the enzymatic modification of triolein to structured lipids

The ability of an immobilized lipase to modify the fatty acid composition of (88.8% C_18:1, 4.3% C_16:0, 3.1% C_18:0, and 3.8% C_18:2 as determined by gas chromatography, and approximately 90% triolein) in hexane by incorporation of a medium-chain fatty acid, capric acid (C₁₀), to form structured triacylglycerol was studied. Response surface methodology was used to evaluate the effect of synthesis variables, such as reaction time (12–36 h), temperature (25–65°C), molar substrate ratio of capric acid to triolein (2:1–6:1), and enzyme amount (10–30% wt% of triacylglycerol), on the yield of structured lipid. Optimization of the transesterification was attempted to obtain maximum yield of structured lipid while using the minimum molar substrate ratio and enzyme amount as much as possible. Computer-generated contour plot interpretation revealed that a relatively high molar substrate ratio (6:1) combined with low enzyme amount (10%) after 30 h of reaction at 25°C gave optimum incorporation of capric acid. A total yield for combined monoand dicaproolein of up to 100% was obtained.     

Phase transformation in CeO2–Co3O4 binary oxide under reduction and calcination pretreatments

The CeO₂–Co₃O₄ binary oxide was prepared by impregnation of the high surface area Co₃O₄ support (S.A. = 100m² g⁻¹) with cerium nitrate (20 wt% cerium loading on Co₃O₄). Pretreatment of CeO₂–Co₃O₄ binary oxide was divided both methods: reduction (under 200 and 400 °C, assigned as CeO₂–Co₃O₄–R200 and CeO₂–Co₃O₄–R400 and calcination (under 350 and 550 °C, assigned as CeO₂–Co₃O₄–C350 and CeO₂–Co₃O₄–C550). The binary oxides were investigated by means of X-ray diffraction (XRD), nitrogen adsorption at −196 °C, infrared (IR), transmission electron microscopy (TEM), diffuse reflectance spectroscopy (DRS) and temperature programmed reduction (TPR). The results showed that the binary oxides pretreatment under low-temperatures possessed larger surface area. The cobalt phase of binary oxides also was transferred upon the treating temperature, i.e., the CeO₂–Co₃O₄–R200 binary oxide exhibited higher surface area (S.A. = 109m² g⁻¹) and the main phase was CeO₂,Co₃O₄ and CoO. While, the CeO₂–Co₃O₄–R400 binary oxide exhibited lower surface area (S.A. = 40m² g⁻¹) and the main phase was CeO₂, CoO and Co. Apparently, the optimized pretreatment of CeO₂–Co₃O₄ binary oxide can control both the phases and surface area.     

GC determination of synthetic hydrocarbon-based thermal heating fluid in vegetable oils

A combination of alumina column chromatography and GC procedures was developed for the determination of synthetic hydrocarbon-based thermal heating fluid (trademarked as Therminol 55^™) in vegetable oils. In each run, 3 g of sample solution was loaded onto the alumina (50 g) column and was eluted with 200 mL hexane. The eluate was then concentrated to 1 mL with the aid of a nitrogen stream prior to GC analysis. The GC chromatogram of the thermal heating fluid was characterized by the presence of a bell-shaped hump that could not be resolved into individual peaks. The lowest detection limit based on various spiked palm olein samples was 20 μg/g. The CV obtained were 6.5–8.9% for the intra-day study and 8.1–9.7% for the inter-day study. Overall recoveries of these samples ranged from 68.0 to 85.7% and the method was found to be good enough for quality control purposes.     

The composition and metabolism of high density lipoprotein subfractions

The composition and metabolism of high density lipoprotein (HDL) subfractions were investigated in seven nomal individuals. Mean HDL₂ (d, 1.063–1.125 g/ml) composition (by weight) was 43% protein, 28% phospholipid, 23% cholesterol, and 6% triglyceride, and mean HDL₃ (d, 1.125–1.21 g/ml) composition was 58% protein, 22% phospholipid, 14% cholesterol, and 5% triglyceride. The mean apoA-I; apoA-II weight ratio was 4.75 for HDL₂ and 3.65 for HDL₃.HDL₂ protein was proportionally slightly richer in C apolipoproteins and higher molecular weight constituents (including apoE) than HDL₃. Kinetic studies utilizing radiolabeled HDL_A (d, 1.09–1.21 g/ml), HDL₂, and HDL₃ demonstrated rapid exchange of apoA-I and apoA-II radioactivity among HDL subfractions, similar fractional rates of catabolism of apoA-I and apoA-II within HDL, and similar radioactivity decay within HDL subfractions. Mean plasma residence time was 5.74 days for radiolabeled HDL₂ and 5.70 days for radiolabeled HDL₃. Differences in HDL protein mass among individuals were largely due to alterations in catabolism, and in general both HDL₂ and HDL₃ were catabolized via a plasma and a nonplasma pathway. Data from simultaneous radiolabeled very low density lipoprotein and HDL studies in 2 individuals are consistent with the concept that apoC-II and apoC-III are catabolized at a different rate than are apoA-I and apoA-II within the HDL density range.     

Rheotechnological properties of mixed suspensions in the SiO2 - Al2O3 system and some properties of materials based on them. 1. Molten quartz – Alumina system

The rheotechnological properties of mixed suspensions in the SiO₂ - Al₂O₃ system obtained by the method of mixing of individual suspensions of molten quartz and alumina are described. Some properties of the materials after their heat treatment at 1000 – 1300°C are investigated. The ranges of compositions (30 – 40% Al₂O₃, 60 – 70% SiO₂) in which the parameters of thermal expansion of the materials are 2 – 3 times lower than those calculated under the condition of additivity are determined. The obtained materials possess an elevated mechanical strength. Translated from Ogneupory i Tekhnicheskaya Keramika, No.7, pp. 18 – 23, July, 2000.     

Identification and structure elucidation of the components of commercial linear alkylbenzenes

A gas chromatography and gas chromatography mass spectrometry study was performed on four commercial linear alkylbenzenes (LAB). A preliminary detailed analysis was done on some model compounds: four tetralin mixtures obtained by alkylation of benzene with linear dichloro n-paraffins (C₁₀, C₁₁, C₁₂, and C₁₃), 1-methyl-4-heptyltetralin and 1-methyl-3-octylindane. We concentrated on the minor components (5–10%) present in the linear alkylbenzenes. Three different types of compounds were identified: A) branched alkylbenzenes, B) 1,4-dialkyltetralins with linear alkyl groups, and C) 1,4-dialkyltetralins with branched alkyl groups. Quantitative evaluation of these minor components was also done. No evidence of 1,3-dialkylindane structures, at least those with linear alkyl groups, was found in the commercial linear alkylbenzenes studied.     

Characterization of feline omentum lipids

Feline omental lipid extracts, previously reported to be angiogenic in the cornea of rabbits, were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1 and 18∶2 fatty acids. Trace quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC)-mass spectrometry, was found to consist of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by high performance thin layer chromatography and HPLC of their perbenzoylated derivatives, were found to consist of glucosyl- and galactosylceramides, galabiosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The complex glycolipid fraction, obtained from Folch upper phase solvent partition, was found to consist primarily of Forssman glycolipid and gangliosides GM₃ and GD₃. Smaller amounts of GM₁ and other unidentified gangliosides were also present. The ganglioside nomenclature is according to the system of Svennerholm (J. Neurochem. 10, 613–623, 1963)     

Sperm oil replacements from selectively hydrogenated soybean and linseed esters: Special lubricants

Soybean and linseed oils were selectively hydroenated with copper-on-silica gel catalyst. The linolenate content of the oils was reduced to diene and monoene with no appreciable increase in saturates. Hydrogenated soybean oils contained 68–76% monoene, 11–18% diene, 0% conjugated diene and triene, 1–6% conjugatable diene, 0–0.3% conjugatable triene, and 23–40% isolatedtrans double bonds. Hydrogenated linseed oils contained 44–54% monoene, 35–45% diene, 0% conjugated diene and triene, 0–7% conjugatable diene, 0–02% conjugatable triene, and 44–59% isoaltedtrans double bonds. Esters of fatty acids, derived from these selectively hydrogenated oils, were prepared with trimethylolethane, trimethylolpropane, trimethylolbutane, pentaerythritol, ethylene glycol, C₁₈ saturated cyclic alcohols, primary C₁₂–C₁₈ saturated (nC₁₂, nC₁₄, nC₁₆, nC₁₈) alcohol, and primary C₁₆–C₁₈ saturated (nC₁₆, nC₁₈) alcohol blends. Measurements of viscosities and of smoke, flash, and fire points indicate that these esters are possible replacements for sperm oil. Certain of them, after sulfurization, also have potential as extreme pressure lubricant additives. Presented at the AOCS meeting in Philadelphia, September 1974.