Rat Serum Lipoproteins Research Articles

Agarose–starch gel electrophoresis of rat serum lipoproteins

Rat serum lipoproteins were separated into at least four fractions by agarose-starch gel electrophoresis. The system used was discontinuous in that glycine and sodium barbitone buffer was used in the reservoirs and Tris buffer was used for the gels. The four major bands could be related to the pattern obtained by ultracentrifugation. The high density lipoproteins consisted of at least two poorly resolved bands and were not separated from albumin. The vertical gel apparatus was further modified to accept 0.4 ml of rat plasma, which was prestained with Sudan black. After electrophoresis the different lipoprotein bands could conveniently be cut out and the lipid phosphorus determined. The addition of Sudan black B decreased the recovery of the low and high density lipoproteins by 5-9%. However, the recovery of phospholipids was reproducible (80 +/- 2%) and the high density lipoproteins contained over two-thirds of the plasma lipid phosphorus.

Isolation and characterization of subunit polypeptides from apoproteins of rat serum lipoprotein

The polypeptide components of the very low density, low density and high density apolipoproteins were fractionated and partially characterized. The fractionation was performed by Sephadex gel filtration and DEAE-cellulose chromatography both in the presence of 8 M urea. Several units were identified: (a) one that was distinctive of low density lipoprotein and also present in very low density lipoprotein; (b) a second one which was the major protein of very low density lipoprotein and a minor component of high density lipoprotein; (c) a third one which was the dominant component of high density lipoprotein; and (d) a group of proteins of low molecular weight, present in small quantities in all lipoprotein classes. All of these polypeptides had distinct characteristics in terms of amino acid composition, disc electrophoretic mobility and immunochemical reactivities.

Activation of lipoprotein lipase. Effects of rat serum lipoprotein fractions and heparin.

Guinea pig serum is deficient in high density lipoproteins (HDL); post-heparin lipoprotein lipase (LPL) from it hydrolyzes a triglyceride (TG) emulsion very slowly. The rate is markedly increased by the addition of rat serum and even further by rat serum plus heparin. We have studied further the activation of LPL in this system. Rat serum lipoprotein fractions were isolated by ultracentrifugation and added to guinea pig postheparin serum in the presence or absence of heparin. When added in proportion to their original serum concentrations, HDL caused the greatest increase in TG hydrolysis. When each fraction was added at equal protein concentrations, purified low density lipoproteins had almost no effect; both very low density lipoproteins and HDL were very effective in increasing the rate of hydrolysis. Rat serum or rat HDL, added to the assay system in increasing amounts, appeared to increase the effective substrate concentration. In the absence of heparin, increasing the concentration of HDL increased the reaction rate which approached a limiting velocity (V max ) and produced a hyperbolic curve which conformed to Michaelis-Menten kinetics. In the presence of heparin, increasing the concentration of HDL produced an S-shaped curve and increased the V max . These data conformed to the sigmoidal kinetics described by the Hill equation. Our results suggest that (1) heparin may function as a specific ligand which acts as an allosteric modifier of LPL and alters the kinetics of interaction of LPL with the effective substrate and (2) the rate of hydrolysis of the effective TG substrate is regulated by the concentration of this substrate.

The dissociation in vitro of the alpha- and beta-lipoprotein components of human and rat very low density lipoproteins.

Heparin precipitates of human and rat serum lipoproteins, dissolved, dialyzed, and analyzed by paper electrophoresis, were found to migrate as β-lipoproteins. The pre-β-lipoproteins disappeared. Using specific anti-human β- and α-lipoprotein sera it was shown that the heparin precipitate of human very low density lipoproteins (VLDL) contained β-lipoproteins, whereas the supernatant contained α-lipoproteins. This indicates that these components are dissociated by the heparin precipitation procedure. Following partial delipidation of the heparin precipitate, a procedure which exposes the antigenic site of the α-lipoprotein of the VLDL, it can be demonstrated that the α-lipoprotein component is completely removed by the heparin precipitation procedure. Partially delipidated rat VLDL showed four immuno-reactive components when reacted against rabbit anti-rat VLDL serum, but only one, corresponding to the β-lipoprotein, when reacted with rabbit antiserum to heparin precipitate.Chemical analysis of the lipid components of the heparin precipitate and supernatant of human and rat VLDL reveals that over 90% of the triglycerides are precipitated by the heparin and that the supernatant fraction contains lipoproteins having a phospholipid to protein ratio higher than any of the other lipoproteins.Upon reincubation of the dissolved and dialyzed human serum heparin precipitate and its supernatant a pre-β band reappeared on paper electrophoresis. The recombined pre-β-lipoprotein was isolated and found to react with anti-β-, but not with anti-α-lipoprotein serum. This is a characteristic of native pre-β-lipoproteins. Evidence has also been obtained that the high density lipoproteins combine only with the β-lipoproteins originally present in the VLDL.

The carbohydrate components of rat serum lipoproteins.

SummaryThe protein of low density rat serum lipoproteins contained 5.2% carbohydrate, consisting of galactose (33%), mannose (31%), glucose (9%), glucosamine (20%), and N-acetyl neuraminic acid (8%). The protein of high density rat serum lipoproteins contained 2.2% carbohydrate, consisting of galactose (23%), mannose (24%), glucose (8%), glucosamine (26%), and N-acetyl neuraminic acid (18%).

Agents affecting lipid metabolism. XXXVII. Separation of rat serum lipoproteins with dextran sulfate.

Dextran sulfate was used to separate rat serum lipoproteins into insoluble low-density and soluble high-density fractions. Lipid composition of the separated fractions was compared to that of lipoprotein fractions prepared by ultracentrifugation; data indicate that dextran sulfate precipitates lipoproteins with a density lower than 1.063.In rats treated with ethyl p-chlorophenoxyisobutyrate and estradiol-17β comparable, though not identical, changes in the composition of serum lipoproteins were observed with both methods of separation. The observed differences may reflect the fundamental difference in the basis of separation used by the methods. Data obtained with dextran sulfate may indicate changes in the protein moieties of lipoproteins.

Human and rat serum proteins, lipoproteins, ammonium persulfate, gel concentration, and disc electrophoresis

Abstract Since the presence of residual ammonium persulfate in polyacrylamide gels has been reported to cause artifacts in electrophoretic patterns of certain proteins, extensive electrophoretic experiments were conducted with human and rat serum proteins and lipoproteins. These experiments included substitution of riboflavin and light as a catalyst for ammonium persulfate at several gel concentrations, removal of ammonium persulfate from the gel by preliminary electrophoresis, and incubation of serum proteins and isolated lipoproteins with ammonium persulfate prior to electrophoresis. The riboflavin catalyzed gels were much softer and were of larger pore sizes than ammonium persulfate catalyzed gels of the same acrylamide concentration and, therefore, the elecerophoretic patterns in the two gel systems were different. However, differences were not observed when the ammonium persulfate was was removed by preliminary electrophoresis. Similarly, incubation of samples with ammonium persulfate did not produce spurious bands and, thus, established that ammonium persulfate does not produce artifacts during disc electrophoresis of serum lipoproteins and proteins using the pH 9.5 system.

Rat serum lipoproteins after parital hepatectomy.

Rat serum lipoproteins after parital hepatectomy.

Serum lipoproteins in rats with tumors induced by 9,10-dimethyl-1,2-benzanthracene and with transplanted Walker carcinosarcoma

Serum lipoproteins in rats with tumors induced by 9,10-dimethyl-1,2-benzanthracene and with transplanted Walker carcinosarcoma

The interaction of ethylene glycol with rat-serum lipoproteins

The interaction of ethylene glycol with rat-serum lipoproteins

Serum lipoproteinemia in pregnant and lactating rats

Serum lipoproteins of pregnant and puerperal rats were studied by preparative and analytical ultracentrifugation. The concentration for the fraction with density less than 1.019 was markedly elevated in rats during the 3rd wk of gestation and in lactating rats. This fraction showed similar triglyceride fatty acid composition and immunoelectrophoretic behavior whether it was derived from pregnant or nonpregnant rats, and when partially delipidized, the lipoproteins from both groups showed similar immunoelectrophoretic characteristics and sedimentation rates. When lactation was interrupted during puerperium, serum lipoproteins returned to control levels; but in lactating rats, high levels of serum very low density lipoprotein persisted up to 3 wk post partum.

The influence of polyacrylamide concentration on human and rat serum lipoprotein patterns

The influence of polyacrylamide concentration on human and rat serum lipoprotein patterns

Disk electrophoresis of isolated rat-serum lipoproteins

1. 1. Disk electrophoresis was performed in 3.75% polyacrylamide gel with ultra-centrifugally isolated rat-serum lipoprotein fractions. These included the very low density, low density and two high density lipoprotein fractions. 2. 2. Four different stains, namely, sudan black B, amido black 10B, oil red O and iodine were employed and the results indicated the presence of at least seven lipo-protein components among the various fractions. Densitometry has been conducted on the gel patterns and has proved useful in the identification of the several disk-electrophoretic components. 3. 3. By comparison of the rat-serum lipoprotein patterns with those obtained with human-serum lipoproteins, it was concluded that the rat low density lipoproteins contained components which were probably smaller in size than those of the human-serum low density lipoproteins. The possibility that one of these components might represent the rat high density lipoprotein component (HDL 1, d = 1.050) has been indicated. 4. 4. The principal component of rat high density lipoprotein (1.125 < d < 1.21) migrated slower than the principal component of the rat high density lipoprotein (1.063 < d < 1.125).

Effect of cold exposure on serum lipids and lipoproteins in the rat.

The effect of cold exposure for 1–52 days on the concentration and composition of the serum lipids and lipoproteins in male Wistar rats was studied. Serum lipoproteins were separated by preparative ultracentrifugation into three density classes, very low density (d &lt; 1.006), low-density (d, 1.006–1.070), and high-density (d, 1.070–1.218). On the first day of cold exposure, total serum lipids were reduced by 20–25%, this decrease occurring mainly in the triglyceride fraction. Although the low-density and high-density lipoproteins were not affected by cold exposure, the concentration of the very low density lipoproteins fell markedly to 70–75% below normal levels. These alterations in the pattern of serum lipids and lipoproteins persisted throughout the cold-exposure period. Sex or prior feeding of a high-fat, high-carbohydrate, or high-protein diet did not modify these cold-induced changes. The response of the very low density lipoproteins to cold was immediate and rapid, the rate and degree of change being greatest in the first 4 hours of exposure. Conversely, the recovery of the very low density fraction to normal after removal of the rats from the cold was slow and depended upon the period of prior exposure. Animals exposed to 4 °C for 4 days required 3 days at 22 °C for the very low density lipoproteins to return to normal, and the recovery period was twice as long for cold-acclimatized (4 weeks) animals. The role of the triglyceride-rich very low density lipoproteins as a readily available energy source in the animals is discussed in the light of these results.

Effect of the cholestrol biosynthesis inhibitor AY-9944 on serum lipoproteins of rats, pigs and dogs.

AY-9944, an orally active inhibitor of cholesterol biosynthesis, caused in rats, pigs and dogs greatly reduced serum cholesterol levels, only partly compensated by 7-dehydrocholesterol formed instead of cholesterol by the action of AY-9944. The distribution of 7-dehydrocholesterol between serum lipoprotein fractions differed from cholesterol and varied from species to species. Levels of 7-dehydrocholesterol and their distribution may reflect the rate of sterol biosynthesis.

Serum lipoproteins in rats with carbon tetrachloride-induced fatty liver

After the administration of CCl4 to male rats, liver triglycerides began to increase after a lag period of about 1 hr; the level of serum triglycerides fell sharply during the first 30 min of intoxication. Three classes of serum lipoproteins were isolated by flotation in the ultracentrifuge and their concentrations and chemical compositions were determined. Within 4 hr of the administration of CCl4 the level of the very low density (VLD-) lipoproteins fell to 25% of that in the control rats. Smaller decreases in the levels of the other two classes of lipoproteins were evident. The serum concentration of all the components of the VLD-lipoproteins were reduced, but proportionally more lipids were bound to the protein moiety in the CCl4-treated rats than in the controls. The concentrations of protein and triglycerides of the VLD-lipoproteins declined most steeply during the first hour of intoxication. The results are interpreted as further evidence that the fatty liver induced by CCl4, is due to a block in the release of hepatic triglycerides to the plasma, the primary lesion being, very probably, inhibition of the synthesis of the protein moiety of serum lipoproteins.

Incorporation of lipoprotein-borne triglycerides by adipose tissue in vitro

Rat adipose tissue was shown to take up triglycerides (TG) upon incubation with isolated human or rat serum lipoproteins. In the physiologic TG concentration range, the uptake in 3 hr was proportional to TG concentration in the medium, without regard to the nature of the TG carrier (lipoproteins of different density classes or chylomicrons). At low TG concentrations an increase in fractional uptake was found. The TG incorporated were found partly in the fat layer and partly dissolved in an aqueous tissue compartment. When doubly labeled TG (fatty acid-C14, glycerol-H3) were used, the TG of the soluble compartment retained the initial C14/H3 ratio of radioactivity, were released in part from the tissue upon reincubation in protein-free medium, and were still contained in intact lipoproteins, immunochemically identical with the original lipoproteins of the medium. On the other hand, the TG in the fat layer had undergone partial transesterification, as inferred from the increase in the ratio of isotope radioactivity. TG elaborated within the tissue by esterification of free fatty acids or by synthesis from glucose were not released into any of several media investigated, so that the release mentioned above does not represent a physiologic mechanism for surrender of tissue fat. It is concluded that incorporation of lipoprotein-borne TG into adipose tissue proceeds in two stages, at first in the intact lipoprotein into a soluble compartment, followed by a shift into the fat droplet, during which some exchange of TG glycerol takes place. The efficiency of the latter stage appeared to determine the over-all rate of uptake of lipoprotein TG, as concluded from kinetic studies of uptake into the two tissue compartments and from the effects of age, nutritional conditions, and temperature. Uptake and partition of TG in the tissue compartments, as well as the extent of transesterification, were also studied in adipose tissues of different anatomic sites of the rat, guinea pig, rabbit, cat, and dog.

EFFECT OF ETHIONINE ON THE INCORPORATION OF PALMITIC ACID-1-C14 INTO RAT SERUM LIPOPROTEINS.

AbstractEthionine administration to female rats had no significant effect on the radioactivity of the serum chylomicron fraction after ingestion of palmitic acid‐1‐C14. On the other hand, ethionine administration resulted in a decrease in the incorporation of palmic‐acid‐1‐C14 into the serum albumin plus α1lipoprotein fraction and into the α2lipoprotein fraction. There was no significant difference in the activity of the serum β lipoprotein fraction between the ethionine and control rats. Total liver fat and the total amount of palmitic acid‐1‐C14 incorporated in the liver fat were significantly higher in the ethionine treated animals than in the saline controls. However, the relative specific activity of the liver fat of the ethionine treated group was lower than that of the controls.

Turnover of rat serum lipoprotein cholesterol.

AbstractKarvinen, E. and M. Miettinen. Turnover of rat serum lipoprotein cholesterol. Acta physiol. scand. 1963. 58. 250–254. — The distribution of cholesterol‐4‐C14 in a1, a2 and β lipoprotein and chylomicron fractîons separated by paper electrophoresis was studied from 3 hours to 8 days after oral administration of cholesterol‐4‐C14. The apparent turnover time of cholesterol‐4‐C14 in the lipoprotein and chylomicron fractions was found to be very similar in all fractions or about 79 hours.

Distribution of ingested palmitic acid-1-C14 between rat serum lipoprotein fractions.

AbstractUptake of palmitic acid‐1‐C14 in albumin plus α1 lipoprotein fraction, in α2 lipoprotein, SbT lipoprotein and chylomicron fractions separated by paper electrophoresis was followed from 3 to 76 hours after oral administration of palmitic acid‐1‐C14. Except the initial high activity in the chylomicrons, the highest activity was found in the albumin plus α1 lipoprotein fraction. Apparent turnover time of this fraction between 3 and 18 hours after ingestion of the label was about 12 hours. Activity of the α2 lipoprotein fraction was not much altered up to 28 hours. Activity of the β lipoprotein fraction seemed to follow the activity of the chylomicron fraction to a greater extent.