Glycosphingolipids of human thyroid

Glycosphingolipids were isolated from total lipids of female and male human thyroids by alkaline hydrolysis, silicic acid, diethylaminoethyl-celluose and thin layer chromatography and analyzed by gas liquid chromatography. On the basis of their mobility in two dimensions on thin layer chromatography, IR analysis, and of sugar molar ratio, four neutral glycolipids, a sulfatide, and a hematoside fraction were identified. Glucosyl, plus galactosyl ceramide, and trihexosyl ceramide were the major fractions and accounted for 33% and 28% of total neutral glycolipids, respectively. Dihexosyl ceramide was a mixture of lactosyl and digalactosyl ceramide. The acidic lower phase glycolipids comprised ceramide galactosyl sulfate as the major component of male thyroids. Hematoside was identified tentatively as a minor component of the thyroids of both sexes. Major fatty acids of all neutral glycolipid fractions were 20∶0, 22∶0, 24∶0, and 24∶1; 24∶0 and 24∶1 for sulfatides. Low proportions of α-hydroxy fatty acids were identified. Total neutral glycosphingolipids of male thyroids were comparable in quantities with human liver but lower than kidneys, leucocytes, and platelets. Male thyroids comprised higher quantities of neutral glycosphingolipids (4.04±0.32 μmoles/g total lipid) as compared to females (2.34±0.21 μmoles/g total lipid), and much higher sulfatide than the females. These marked differences may suggest that the biosynthesis of the glycosphingolipids in the thyroid gland is under hormonal control. Similarities in glycosphingolipid composition of human thyroid and kidney are discussed in relation to a possible role played by glycolipids in ion transport, which is a common feature of the two organs.     

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.     

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.     

Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins

Previous observations demonstrated that steroid hormones associate with plasma lipoproteins. The objective of this study was to estimate the relative importance of lipoproteins as steroid hormone binding agents in comparison to sex hormone binding globulin, corticosteroid binding globulin, and albumin in both normal and hyperlipidemic human plasma. The 16 steroid hormones and related metabolites included in the study were: androstanediol, androstenediol, androstenedione, androsterone, corticosterone, cortisol, dehydroepiandrosterone, deoxycorticosterone, dihydrotestosterone, estradiol, estriol, estrone, 17α-hydroxyprogesterone, pregnenolone, progesterone, and testosterone. The binding activity of these 16 steroid hormones with purified high density lipoprotein (HDL), low density lipoprotein and very low density lipoprotein were separately evaluated by equilibrium dialysis incubations to yield 48 steroid hormone-lipoprotein combinations for further study. In incubations with HDL, six steroid hormones (androstenediol, dehydroepiandrosterone, dihydrotestosterone, estradiol, pregnenolone, and progesterone) were identified as non-equilibrium, apparently due to metabolic conversion of the steroid hormones. The metabolic activity for the three Δ⁵-3β hydroxy steroids and estradiol appears to be fatty acid esterification by lecithin:cholesterol acyltransferase. The computer program TRANSPORT, which was used to evaluate only the nonspecific steroid hormone-lipoprotein association levels in a 16×6 matrix at simultaneous equilibrium, indicated that lipoprotein-bound steroid hormones ranged from 1% for cortisol to 56% for pregnenolone in normal human blood. Simulated projections of the increase in nonspecific steroid hormone association with lipoproteins during hyperlipidemia are also presented. These results demonstrate how lipoproteins are likely to be important in the transport and metabolism of steroid hormones in human plasma.     

High performance reversed phase chromatography of cholesterol and cholesteryl esters of human plasma lipoproteins

Cholesterol and cholesteryl esters were separated according to their carbon number and number of double bonds by high performance reversed-phase chromatography (HPRC) using acetonitrile/chloroform/methanol (1∶1∶1, v/v) as a mobile phase. It was found that within the same equivalent carbon number (ECN) category, cholesterol esters with the highest number of double bonds eluted ahead of those with a lower number of double bonds, and with thecis isomers eluting ahead of theirtrans partners. Thus, cholesteryl oleate (C27-18∶1c) elutes ahead of cholesteryl palmitate (C27-16∶0) and ahead of cholesteryl elaidate (C27-18∶1t). Human lipoprotein, as well as rat liver cholesteryl esters, were separated using this technique.     

Neutral lipid transfer protein does not regulate α-tocopherol transfer between human plasma lipoproteins

Vitamin E has no known plasma carrier protein and is transported by plasma lipoproteins. The site of association of vitamin E in the lipoprotein particle and the mode of transfer of vitamin E between plasma lipoproteins have not been ascertained. Since neutral lipids (triglycerides and cholesterol esters) exchange between plasma lipoproteins by processes mediated by neutral lipid transfer protein, we questioned that if vitamin E, a hydrophobic molecule, is carried in the core of the lipoprotein particle then its transfer between plasma lipoproteins may be mediated by neutral lipid transfer protein. Transfer of D-α(5-methyl-³H)tocopherol from in vitro-labeled human plasma lipoprotein fractions to other plasma lipoproteins was measured under incubation conditions that were designed to yield markedly differing degrees of neutral lipid exchange. Despite the presence of the d>1.21 g/ml lipoprotein-poor plasma fraction or purified lipid transfer protein that resulted in up to a 10-fold increase in neutral lipid transfer, vitamin E transfer between very low density lipoproteins, low density and high density lipoproteins remained constant. Even excess amounts of lipid transfer protein, which caused triglyceride transfer between very low density and high density lipoproteins to reach saturation, failed to affect significantly vitamin E transfer. Vitamin E distribution between lipoprotein fractions did correlate with lipoprotein mass ratios. Vitamin E transfer was higher as the protein ratio of acceptor lipoproteins to donor lipoproteins increased. We conclude that vitamin E transfer between lipoproteins is not dependent primarily on neutral lipid transfer protein and is not mediated via neutral lipid transfer.     

The interaction of human plasma low density lipoproteins with glycosamino-glycans: Influence of the chemical composition

Human plasma of 5 normolipemic individuals was incubated for 24 hr at 37 C in the presence or in the absence of lecithin: cholesterol acyltransferase (LCAT)-inhibitors. Plasma steored at 4 C served as a control. The low density lipoprotein (LDL) fractions of the samples were isolated and investigated with respected to changes in chemical composition and complexing activity with glycosamino glycans (GAG). Incubation of plasma in the presence of LCAT inhibitors caused a significant increase of LDL triglycerides at the expense of cholesteryl esters. Incubation with active LCAT not only changed the core but also the surface constituents (decrease in phospholipids and in free cholesterol). The amount of GAG bound per mg of LDL was not uniformly changed in samples incubated after LCAT inhibition. LDL isolated from plasma incubated in the presence of LCAT, on the other hand, showed a significant reduction in GAG binding. The ratio of free cholesterol: GAG in the complex was most significantly reduced in LCAT-modified LDL. There was in addition a highly significant correlation between the LDL:GAG ratio in the complex and the free cholesterol and phospholipid content of the LDL samples. It is concluded that alterations in surface lipid constitutents of LDL strongly after their interaction with sulfated polysaccharides, an effect which may be relevant also in vivo for the interaction of LDL with cell surfaces and intercellular matrices.     

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.     

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.     

Net lipid transfer between lipoproteins in fish-eye disease plasma supplemented with normal high density lipoproteins

Native fish-eye disease plasma, which is deficient of both high density lipoproteins (HDL) and lecithin-cholesterol acyltransferase activity (α-LCAT), processing the free cholesterol of these lipoproteins, has been supplemented with normal isolated HDL₂ or HDL₃ and incubated in vitro at 37 C. After incubation for 0,7.5 and 24 hr the very low density (VLDL) and low density (LDL) lipoproteins as well as HDL were isolated, and their contents of triglycerides, phospholipids and free, esterified and total cholesterol were quantified. The resulting net mass transfer of the different lipids revealed a functioning transfer of cholesteryl esters and all other analyzed lipids between the lipoproteins, although no de novo esterification of the HDL cholesterol by LCAT in this plasma occurred. In accordance with previous findings there was a functioning esterification process of the free cholesterol of the combined VLDL and LDL of fish-eye disease plasma. The present results make it reasonable to conclude that the lack of HDL cholesterol esterification in this disease is not a result of a deficiency of cholesteryl ester transfer or lipid transfer activities.     

Properties of ultrasonically irradiated human serum lipoproteins

Human serum low density lipoprotein (LDL) and human serum high density lipoprotein (HDL) were treated with ultrasonic irradiation. The immunochemical properties, spectrophotometrical analysis and thiobarbituric acid test (TBA) value of ultrasonically irradiated lipoproteins were examined. The agar gel precipitin reaction of sonicated LDL disappeared as the irradiation time increased. The effects of ultrasonic irradiation upon human serum lipoproteins resulted in a loss of lipids from the sonicated lipoproteins and are increased in TBA value. TBA value of LDL increased in two steps.     

Effects of diet and type IIa hyperlipoproteinemia upon structure of triacylglycerols and phosphatidyl cholines from human plasma lipoproteins

Four normal and two individuals with Type IIa hyperlipoproteinemia were placed on the National Heart and Lung Institute Type IIa diet (low cholesterol, smaller than 300 mg/day, high polyunsaturated, low saturated fat diet) for 1 week and on a normal diet the following week. Plasma samples were obtained and the triacylglycerols, phospholipids, and cholesterol contents of plasma and of very low density lipoproteins, low density lipoproteins, and high density lipoproteins determined. Triacyglycerol fatty acid composition was determined and stereospecific analyses of triacglycerols and phosphatidyl cholines performed. Structural determinations were limited to one normal and one Type IIa individual. In normal and Type IIa individuals, chylomicrons contained twice the amount of 18:0 as did the very low density lipoproteins, low density lipoproteins, or high density lipoproteins. The structure of the triacyglycerols from the very low density lipoproteins and low density lipoproteins was asymmetric with at least 50M% 16:0 in the sn-1 position and mostly 18:1 in positions sn-2 and 3. There was a marked difference in the distribution of 18:2 in low density lipoproteins of the normal and Type IIa individuals. The control contained equal amounts of 18:2 in the sn-1 and sn-3 positions, whereas IIa low density lipoprotein was asymmetric with 26% of the 18:2 in position sn-1 and 3% in the sn-3 position. Very low density lipoprotein was asymmetric with regard to 18:2 in control and IIa samples with an average of 5% of the 18:2 in position sn-1 and 40% in position sn-3. The phosphatidyl cholines contained predominantly 16:0 and 18:0 in position sn-1, whereas the acids in position sn-2 were unsaturated with very little difference between lipoprotein classes. Neither the short dietary periods nor source of plasma affected the structure of the phosphatidyl cholines.     

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.     

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.     

Tocopherol contents of lipoproteins from frozen plasma separated by affinity chromatography

We investigated whether freezing and storage of plasma altered α-tocopherol levels of whole plasma or the lipoprotein fractions derived from such plasma. Plasma from 24 men, at each of two collection periods, was frozen at −20°C for six weeks, then high-density lipoproteins (HDL) were separated from low- plus very low-density lipoproteins (LDL-VLDL) by heparin affinity chromatography. Whole plasma and the lipoprotein fractions were analyzed for α-tocopherol content and compared to counterparts from fresh plasma. Freezing and storage did not reduce α-tocopherol levels of plasma or the lipoprotein fractions. α-Tocopherol values from fresh and frozen plasma were highly correlated for both plasma (period 1, r=0.94; period 2, r=0.93) and the LDL-VLDL fractions (periods 1 and 2, r=0.97). Percent distribution of α-tocopherol between the two lipoprotein fractions was comparable for lipoproteins derived from fresh and frozen plasma. Under the storage conditions used in this study, plasma can be frozen for at least six weeks prior to lipoprotein fractionation with no detectable detrimental effects on α-tocopherol content of either plasma or lipoproteins.     

Effects of stearic acid on plasma lipid and lipoproteins in humans

More than 40 years ago, saturated FA with 12, 14, and 16 carbon atoms (lauric acid, myristic acid, and palmitic acid) were demonstrated to be “hypercholesterolemic saturated FA.” It was further concluded that the serum total cholesterol level would hardly be changed by isocaloric replacement of stearic acid (18∶0) by oleic acid (cis-18∶1n−9) or carbohydrates. These earlier studies did not address the effects of the various FA on the serum lipoprotein profile. Later studies found that the hypercholesterolemic saturated FA increase serum total cholesterol levels by raising concentrations of both the atherogenic LDL and the antiatherogenic HDL. Consequently, the ratio of total to HDL cholesterol will hardly change when carbohydrates replace these saturated FA. Compared with other saturated FA, stearic acid lowers LDL cholesterol. Studies on the effects on HDL cholesterol are less conclusive. In some, the effects on HDL cholesterol were comparable to those of palmitic acid, oleic acid, and linoleic acid, whereas in others a decrease was observed. This may suggest that in this respect the source of stearic acid is of importance, which needs however further study. From all these studies, however, it can be concluded that stearic acid may decrease the ratio of total to HDL cholesterol slightly when compared with palmitic or myristic acid. Without doubt, the effects of stearic acid are more favorable than those of trans monounsaturated FA.     

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.     

Evaluation of the rapid micromethod for ultracentrifugal separation of labeled plasma lipoproteins

The fractionations of plasma lipoproteins by 2 methods were compared to evaluate the rapid separation (Airfuge^®) method for lipoprotein distribution studies. When [¹²⁵I] labeled very low density, low density, and high density lipoproteins (VLDL, LDL, HDL), were separately centrifuged in buffers at d=1.006, 1.06 or 1.2 g/ml by the conventional ultracentrifuge and the Airfuge^®, separations of the fractions in the Airfuge^® were incomplete at both 5 C and 24 C, especially at d=1.006. [³H] Benzo (a)pyrene, when added to plasma, associates with the plasma proteins and lipoproteins, especially LDL. Compared to the standard techniques, the Airfuge^® method greatly overestimated its distribution into VLDL. The distribution of [³H] vitamin D₃ into the VLDL plus LDL fraction was also overestimated by the Airfuge^® procedure. It is concluded that caution should be observed in quantitative studies of lipoproteins in the Airfuge^®. A careful comparison of the distribution into or fractionation of lipoproteins by the 2 methods should always precede any quantitative determinations involving the Airfuge^®.