A rapid method for separation of plasma low and high density lipoproteins for tocopherol and carotenoid analyses

Ultracentrifugation (UC) is the method most often employed for separation and quantification of lipoproteins. Because this procedure requires expensive laboratory equipment, a large volume of fresh sample and an inordinate amount of time, it may not be ideal for routine clinical/experimental use. The aim of the current study was to evaluate a method which combines selective precipitation (HDL-P) and immunoseparation (LDL-I) for the rapid and reliable isolation of high density lipoproteins (HDL) and low density lipoproteins (LDL) specifically for vitamin E and carotenoid determination within these fractions. Cholesterol and triacylglycerol concentrations within the HDL and LDL were also determined to enable expression of vitamin E and carotenoid concentrations per gram of lipid. Isolation of lipoproteins by UC was used as the reference method (HDL-UC/LDL-UC). There were no significant differences between methods for α-and γ-tocopherol in LDL and HDL. Carotenoids measured in HDL and LDL were comparable between the methods. The exception was higher lutein/zeaxanthin concentration in HDL-P and LDL-I compared to HDL-UC and LDL-UC, respectively. Additionally, lycopene concentration was significantly lower in LDL-I compared to LDL-UC. In comparing vitamin E and carotenoid values in lipoproteins separated from fresh and frozen plasma by the direct method, there was no difference in α-tocopherol or the majority of carotenoids measured. In conclusion, a combination of selective precipitation and immunoseparation of fresh or frozen plasma for subsequent α-and γ-tocopherol analyses provides an accurate and reliable alternative to lipoprotein separation by UC. Additionally, carotenoid concentrations in HDL separated by selective precipitation and analyses of α-and β-carotenes and β-cryptoxanthin in LDL separated by immunoseparation are also reliable, while lycopene and lutein/zeaxanthin concentrations in LDL-I are not readily comparable to LDL-UC.     

A rapid micromethod for the quantitative analysis of jojoba wax and its components

A method for the routine determination of jojoba wax ester composition and the ethanolysis products of these esters is described. In the recommended procedure, single or half seeds are crushed onto filter paper disks to provide duplicate 10–20 mg samples of the wax. One paper is extracted with petroleum ether for wax ester analysis and the second sample is transesterified in a sealed bottle using 5% HCl in ethanol at 80 C for 1–2 hr. This preparation is extracted with NaCl and petroleum ether, neutralized with potassium bicarbonate and dried with anhydrous sodium sulfate. The fatty acid ethyl esters and free alcohols are determined by gas chromatography (GC). The method requires only small amounts of seed material, provides duplicate samples of the wax, simplifies the ethanolysis procedures and reduces the time needed for the removal of the acid catalyst.     

A simple,rapid micromethod for the determination of the structure of unsaturated fatty acids via ozonolysis

A micromethod for the localization of double bonds in unsaturated fatty acids via ozonolysis employing pyrolytic cleavage of ozonides in the presence of a hydrogenation catalyst is described. Cleavage of the ozonides is carried out in a gasliquid chromatographie instrument in a small glass tube, containing the catalyst, inserted in the top of the column opposite the in input heaters at 225C. Ozonides of methyl esters of straight chain unsaturated fatty acids are cleaved through the action of the catalyst to aldehyde fragments which are swept simultaneously into the column for analysis. The double bond positions are deduced from the chain length of the fragments. The method is demonstrated on methyl oleate, linoleate, linolenate and arachidonate. Presented at the AOCS Meeting, Cincinnati, October, 1965.     

Glycosphingolipids of human plasma lipoproteins

The content and structure of glycosphingolipids (GSL) in human plasma lipoproteins were studies. The quantitative distribution of the neutral GSL(Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer) and the principal ganglioside (AcNeu-Gal-Glc-Cer) within the different lipoprotein classes was similar to that of whole plasma. The total amounts (μmol glucose/100 ml plasma) of GSL in the plasma lipoproteins of three normal subjects were VLDL (very low density lipoproteins) (trace to 0.46), LDL (low density lipoproteins) (1.08–1.48), HDL₂ (high density lipoproteins₂) (0.62–0.85), and HDL₃ (high density lipoproteins₃) (trace to 0.28). In subjects with Lp(a) lipoproteins, HDL₂ rather than HDL₃ contained most of the GSL in HDL. When the data were corrected for differences in the plasma concentrations of the lipoproteins, the total amounts of GSL(nmol glucose/mg lipoprotein cholesterol) were VLDL(trace to 21.20), LDL(11.70–15.36), HDL₂(8.50–9.10), and HDL₃(3.12). No GSL were detected in lipoprotein deficient plasma. Mass spectrometry of the trimethylsilyl derivatives of the GSL in LDL showed major fragment ions characteristic of their individual structural components. The elevated plasma levels of the GSL(2–18 fold), in a homozygote for familial hypercholesterolemia, resided in LDL which contained an absolute increase (per mg lipoprotein cholesterol) of GSL. Most, if not all, of the plasma GSL are associated with plasma lipoproteins and may have an important role in their biological functions.     

Characterization of plasma lipoproteins in swine with different propensities for obesity

Yorkshire (lean) and Ossabaw (obese) swine ca. one year of age were used to characterize the quantity and composition of plasma lipoproteins in animals with markedly different adiposity. While lean swine weighed more (175 vs 88 kg for obese), they had less backfat than obese swine (2.64 vs 5.97 cm; P<0.05). Fasting plasma triacylglycerol (Tg) and cholesterol (CH) levels were elevated in obese swine. Swine plasma lipoproteins were fractionated into very low density lipoprotein (VLDL; d<1.006), low density lipoprotein₁ (LDL₁; d=1.019–1.063), low density lipoprotein₂ (LDL₂; d=1.063–1.09), and high density lipoprotein (HDL; d=1.09–1.21) by density ultracentrifugation. Obese VLDL-Tg, CH and protein (Pr) were elevated more than 2-fold. VLDL from obese swine were 2-fold larger than VLDL from lean swine. No alterations in LDL₁ or LDL₂ composition were observed. HDL-Tg, CH, Pr and phospholipid levels were significantly higher in obese swine. Plasma and VLDL-Tg levels were highly correlated with backfat thickness (r=0.67 and r=0.73, respectively). These was a positive correlation between adiposity and HDL-CH as well as VLDL-Tg and HDL-CH. These data indicate that (a) there are marked alterations in swine plasma lipoprotein composition between lean and obese swine; (b) that swine plasma lipoprotein levels may be useful parameters in estimating body composition; and (c) that HDL-CH is positively correlated with adiposity in swine. Department of Dairy and Animal Science, College of Agriculture. Nutrition Program, College of Human Development.     

A micromethod for the estimation of blood dolichol

A micromethod for the estimation of dolichol in blood was developed using high performance liquid chromatography. This method can be applied to whole blood or plasma. With detector sensitivity set at 0.005 Absorbance Unit Full Scale, samples as little as 50 μl are sufficient to carry out the determination. Blood samples from the tail vein of inbred strains of C57 BL/6 NNia-1 mice 3,6, 12 and 24 mo of age were examined. Blood dolichol levels decreased with age from 163.3 ng/ml at 6 mo to 110.1 ng/ml at 24 mo. The major dolichol homologs were C-85 (10.4%), C-90 (41.6%), C-95 (38.0%) and C-100 (8.1%).     

A micromethod for the stereospecific determination of triglyceride structure

Triglyceride lipase and diglyceride kinase can be used in a sensitive stereospecific analysis of the separate fatty acid compositions at the 1, 2 and 3 positions of a triglyceride. Diglyceride kinase fromEscherichia coli selectively catalyzes the phosphorylation of 1,2-diglycerides but not the 2,3-diglycerides. The composition of the 3-position in rat liver triglycerides is clearly different from that at the 1-position. A preliminary report of this work has been presented (1).     

High performance reversed-phase chromatography of the triglycerides from human plasma lipoproteins

The triglycerides of human plasma lipoproteins were separated with high performance reversed-phase liquid chromatography. An octadecyl bonded 5-μ silica column was used with a mobile phase of acetonitrile/acetone. Individual triglyceride types and critical pairs may be easily separated and identified.     

L-carnitine effect on plasma lipoproteins of hyperlipidemic fat-loaded rats

The effect of oral L-carnitine administration to rats fed olive oil has been studied. Carnitine significantly decreased triglyceride, cholesterol and phospholipid levels. Particularly, the levels of chylomicron and very low density lipoproteins in the blood were lowered. Low density lipoprotein levels were not affected, and high density lipoproteins were found to be decreased by 20%. Because carnitine did not change the composition of chylomicron and very low density lipoproteins fraction or affect the gastrointestinal triglyceride residue (about 1/3 of the original load), an effect of carnitine on hepatic fatty acid handling is most likely. The lowering of plasma free fatty acid levels by carnitine administration is in favor of an effect of carnitine on fatty acid handling. The effect on the liver is illustrated by the study of acetoacetate formation in in vitro perfused livers from previously olive oil loaded±carnitine-treated rats. Carnitine pretreatment stimulated ketogenesis. It is speculated that carnitine administration, by promoting β-oxidation, lowers the production of very low density lipoproteins. This may be accomplished partly by an increase in the hepatic level of fatty acid binding protein, which also has been observed.     

The effect of exercise on plasma high density lipoproteins

The influence of vigorous activity in man on plasma lipids and lipoproteins is reviewed, with particular emphasis on high density lipoproteins. Both cross sectional and longitudinal (or training) studies have been reported, many of them of less than ideal design. Nonetheless, a consistent pattern emerges in which increased exercise levels lead to lower plasma concentrations of triglycerides and very low density lipoproteins, and of low density lipoproteins. High density lipoprotein levels increase. Sometimes, but not uniformly, plasma total cholesterol level falls as the result of these changes. The increase in plasma high density lipoprotein appears to be the result largely of an increase in the less dense HDL₂ subfraction. Plasma apolipoprotein A-I levels (but not apo-A-II levels) seem to increase concomitantly. The precise biochemical mechanism responsible for these changes has not been elucidated; but the recent finding of increased lipoprotein lipase activity in adipose tissue and muscle of endurance runners suggests that increased lipolytic rate of trigly ceride-rich lipoproteins may be an initial step in a sequence of events leading to higher plasma levels of HDL₂.     

Evidence for the lipoprotein heterogeneity of human plasma high density lipoproteins isolated by three different procedures

Quantitative electroimmunoassays of apolipoproteins in ultracentrifugally isolated high density lipoproteins (HDL) of normolipidemic subjects showed that A-I and A-II are the major (80–85% of total HDL protein) and B, C-III, E, D and F are the minor protein constituents of this density class. A comparison between the apolipoprotein composition of ultracentrifugally isolated HDL and heparin-Mn⁺⁺ supernates showed no significant difference in the levels of A-I and C-III. However, the concentration of ApoE in the heparin-Mn⁺⁺ supernates was almost twice as high as that in the ultracentrifugally isolated HDL; ApoB was only detectable in trace amounts in the heparin-Mn⁺⁺ supernates. To establish whether these apolipoproteins are parts of a single macromolecular complex or form separate, discrete lipoprotein particles, the high density lipoproteins were isolated by three different procedures including ultracentrifugation, heparin-Mn⁺⁺ precipitation and agarose column chromatography. The double diffusion analyses of each of these HDL preparations with antisera to A-I, A-II, ApoB, C-III, ApoD, ApoE, and ApoF showed nonidentity reactions between each possible combination of these antisera. The only exception was a reaction of partial identity between antisera to A-I and A-II polypeptides indicating the occurrence of two types of lipoprotein particles, a major one (LP-A) containing both polypeptides and a minor one (LP-A-I) containing A-I as the sole protein constituent. These findings indicate that high density lipoproteins, regardless of the manner of isolation, do not consist of a single macromolecular complex, but represent a mixture of several, discrete lipoprotein families. Presented at the AOCS Meeting, St. Louis, May 1978.     

Distribution of antithrombin III and glucosylceramide in human plasma lipoproteins and lipoprotein deficient plasma

We have investigated the distribution of antithrombin-III and glucosylceramide (Glc-Cer) in human plasma, plasma lipoproteins and lipoprotein-deficient plasma. Antithrom bin III activity was measured employing immunochemical and biological assays. Glc-Cer was quantified by gas liquid chromatography (GLC). Whole plasma contained 145 μg antithrombin III/ml plasma, all of which was associated with the lipoprotein-deficient plasma (d>1.25 g/ml). Whereas, most if not all the plasma GlcCer was associated with plasma low density lipoproteins (LDL) (d-1.022–1.055 g/ml) and high density lipoproteins (HDL) (d-1.063–1.25). GlcCer was not found in the lipoprotein-deficient plasma. We conclude that GlcCer on lipoproteins does not contribute to antithrombin III activity. Moreover, the absence of GlcCer in lipoprotein-deficient plasma does not impair antithrombin-III activity.     

Porous dye affinity beads for albumin separation from human plasma

Porous polymeric beads were obtained by the suspension polymerization of 2‐hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA). Poly(HEMA–EGDMA) beads were characterized by surfacearea measurements, swelling studies, FTIR, scanning electron microscopy (SEM), and elemental analysis. Poly (HEMA–EGDMA) beads had a specific surface area of 56 m²/g. SEM observations showed that the poly(HEMA–EGDMA) beads abounded macropores. Poly(HEMA–EGDMA) beads with a swelling ratio of 55%, and containing different amounts of Reactive Red 120 (9.2–39.8 μmol/g) were used in the adsorption/desorption of human serum albumin (HSA) from aqueous solutions and human plasma. The nonspecific adsorption of HSA was very low (0.2 mg/g). The maximum HSA adsorption amount from aqueous solution in phosphate buffer was 60.1 mg/g at pH 5.0. Higher HSA adsorption value was obtained from human plasma (up to 95.7 mg/g) with a purity of 88%. The equilibrium monolayer adsorption amount, Q_max was determined as 172.4 mg/g. The dimensionless separation factor (R_L) value shows that the adsorption behavior of HSA onto the Reactive Red 120 attached poly(HEMA–EGDMA) beads was favorable (0 < R_L < 1). Desorption of HSA from Reactive Red 120 attached poly (HEMA–EGDMA) beads was performed using 0.1M Tris/HCl buffer containing 0.5M NaCl. It was observed that HSA could be repeatedly adsorbed and desorbed with Reactive Red 120‐attached poly(HEMA–EGDMA) beads without significant loss in the adsorption amount. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Incubation of lipid emulsions with plasma lipoproteins modifies the fluidity of each particle

Lipid emulsions (LE) contain triglyceride (TG)-rich particles (TGRP) and phospholipid-rich particles (PLRP). Various lipid and protein exchanges take place during in vitro incubations of LE with lipoproteins. These composition changes affect physical properties of particles. The aim of this study was to determine the role of different LE particles and the effect of TG composition on physical modifications. Low density lipoproteins (LDL: 1.025<d<1.040 g/mL) or high density lipoproteins (HDL: 1.085<d<1.150 g/mL) were incubated with the following four LE or their TGRP or PLRP, which were manufactured with the same phospholipid emulsifier: long-chain triglycerides (LCT): 100% soybean oil; medium-chain triglycerides (MCT)/LCT (MCT/LCT, 5∶5, w/w); FO (100% fish oil); and MLF541 (MCT/LCT/FO, 5∶4∶1, by wt). After incubation, modified LE particles and lipoproteins were analyzed by fluorescence polarization. Observed physical modifications were significant in emulsion particles (ordering effect) but not in lipoporteins and also were significant for TG composition effect. Since intact emulsion contained a large excess of TGRP over PLRP, it is not surprising that intact emulsion had the same behavior as TGRP alone, and that PLRP had the same physical characteristics as lipoproteins. TG loss and cholesterol and protein acquisitions by emulsion particles rigidify their envelope. The two emulsions containing FO were less ordered after incubation. In conclusion, incubation of LE with lipoproteins changes physical properties of each kind of particle, and TG composition of the emulsion affects emulsion particle changes but has no effect on LDL and HDL. These order modifications induce more effective exchanges between LE particles and lipoproteins and modify their metabolism; HDL changes may increase the reverse cholesterol transport.     

Polarization-interference micromethod for the study of mutual diffusion in polymer systems

Summary A polarization-interference technique is developed for the study of mutual diffusion that is applicable to systems covering the whole concentration range from polymer — low-molecular-weight compound systems to polymer — oligomer systems. The technique is based on variations of the refraction index within the diffusion zone and is shown to have advantages over similar ones. Its accuracy in terms of wavelength is 0.04. The polybutadiene — dioctyl phtalate system is used to illustrate how the concentration distribution of the components is calculated.     

A simplified colorimetric micromethod for determination of serum cholesterol

A simple and rapid micromethod is described for quantitation of cholesterol in 5–10 μl of serum using uranyl acetate to deproteinize the serum ando-phthalaldehyde reagent to develop color. Independent analyses of serum samples by the present method and by two reference methods, Abell-Kendall and automated colorimetric method, indicated accuracy of the micromethod (r=0.99). Use of capillary blood specimens makes this procedure applicable for screening infants and small children.     

脊髓灰质炎病毒颗粒分离纯化方法学评价

目的比较3种不同密度梯度离心方法对脊髓灰质炎病毒空心颗粒、实心颗粒的分离效果。方法选择蔗糖密度梯度离心、氯化铯密度梯度离心、Optiprep(碘克沙醇溶液)密度梯度离心方法,对脊髓灰质炎病毒进行纯化,抽取离心之后的蛋白条带,通过计算蛋白回收率、SDS-PAGE分析、病毒颗粒透射电镜观察,评价其对病毒空心颗粒、实心颗粒的分离效果。结果病毒液经蔗糖、氯化铯和Optiprep 3种介质超速离心,均能分离空心颗粒和实心颗粒。氯化铯密度梯度离心法蛋白回收率优于Optiprep及蔗糖密度梯度离心法;Optiprep密度梯度离心法蛋白纯度优于氯化铯及蔗糖密度梯度离心法。结论通过蛋白回收率、SDS-PAGE分析、病毒颗粒透射电镜观察初步评价了3种方法对脊髓灰质炎病毒空心颗粒和实心颗粒的分离效果,为脊髓灰质炎病毒分离纯化提供了参考。     

Effects of 17β-estradiol and starvation on trout plasma lipoproteins

Administering 17β-estradiol (E₂) to juvenile trout results in plasma hyperlipidemia and hyperlipoproteinemia associated with significant increases in the concentrations of triglycerides (TG), free cholesterol, phospholipids, free fatty acids and proteins, both postprandial and during starvation. TG undergo the greatest increase (9 times control level 96 h after feeding). The concentration differences between E₂-treated and control trout increase during starvation, primarily by progressive decreases in the concentrations of various lipids in controls. E₂-induced hypertriglyceridemia is mainly caused by an increase in the concentration of very low density lipoproteins (VLDL) during both the postprandial period (6 times control level at 24 h) and during starvation (15 times control level at 96 h); hyperlipoproteinemia lasts up to at least 7 d after the last feeding. E₂ treatment does not change the concentration of high density lipoproteins, but does increase plasma concentrations of a very high density lipoprotein, vitellogenin (VTG). In E₂-treated VLDL, cholesteryl esters are depleted while proteins are enriched. During the postprandial phase, the apolipoprotein (apo) profile of VLDL (d< 1.015 g/mL) is comparable in E₂-treated and control trout. Starvation of E₂-treated trout is accompanied by an enrichment in apo B₂₄₀, A-I and A-II. The secretion levels of TG and VLDL-TG, as determinedin vivo, by injecting Triton WR-1339 to starving animals, are significantly higher in E₂-treated trout than in controls. In trout, as in chicks, E₂ administration significantly increases the concentration and hepatic secretion of plasma VLDL independent of the nutritional status and the appearance of VTG in the plasma. This suggests the existence of similar mechanisms for the regulation of lipoprotein metabolism by estrogens in oviparous vertebrates.     

Inhibition of rat hepatic sterol formation from squalene by plasma lipoproteins

The conversion of³H-squalene to sterols by rat liver microsomes and cytosol was inhibited by individual rat and human plasma lipoproteins at various concentrations. This inhibition was also observed with added human high density apolipoprotein, but triglycerides, cholesterol or cholesteryl esters had no inhibitory effects. Lipoproteins and apo high density lipoprotein (HDL) were demonstrated to bind³H-squalene in vitro. The binding of³H-squalene by apo HDL could be reversed by increasing concentration of liver cytosol containing sterol carrier protein.