Compensatory mechanisms governing the concentration of plasma low density lipoprotein

To evaluate factors regulating the concentrations of plasma low density lipoproteins (LDL), apolipoprotein B metabolism was studied in nine Pima Indians (25 +/- 2 yr, 191 +/- 20% ideal wt) with low LDL cholesterol (77 +/- 7 mg/dl) and apoB (60 +/- 4 mg/dl) and in eight age- and weight-matched Caucasians with similar very low density lipoprotein (VLDL) concentrations, but higher LDL (cholesterol = 104 +/- 18; apoB = 82 +/- 10; P less than 0.05). Subjects received autologous 131I-labeled VLDL and 125I-labeled LDL, and specific activities of VLDL-apoB, intermediate density lipoprotein (IDL)-apoB, and LDL-apoB were analyzed using a multicompartmental model. Synthesis of LDL-apoB was similar (1224 +/- 87 mg/d in Pimas vs 1218 +/- 118 mg/d in Caucasians) but in Pimas the fractional catabolic rate (FCR) for LDL-apoB was higher (0.48 +/- 0.02 vs 0.39 +/- 0.04 d-1, P less than 0.05). In the Pimas, a much higher proportion of VLDL-apoB was catabolized without conversion to LDL (47 +/- 3 vs 30 +/- 5%, P less than 0.01). When all subjects were considered together, LDL-apoB concentrations were negatively correlated with both FCR for LDL-apoB (r = -0.79, P less than 0.0001) and the non-LDL pathway (r = -0.43, P less than 0.05). Also, the direct removal (non-LDL) path was correlated with VLDL-apoB production (r = 0.49, P = 0.03), and the direct removal pathway and FCR for LDL-apoB were correlated (r = 0.49, P = 0.03). In conclusion, plasma LDL appear to be regulated by both the catabolism of LDL and the extent of metabolism of VLDL without conversion to LDL; both of these processes may be mediated by the apoB/E receptor, and appear to increase in response to increasing VLDL production.     

The isolation of lipoproteins from human plasma by ultracentrifugation in zonal rotors

The major classes of lipoproteins were isolated from human plasma by ultracentrifugation in continuous density gradients using the Ti-14 and Ti-15 zonal rotors. Chylomicrons + VLDL, LDL, and HDL were separated from each other and from the more dense residual proteins (albumin fraction) of plasma by rate-zonal flotation in NaBr gradients in the density range 1.0-1.4. The chylomicron-VLDL fraction was subfractionated into constituent chylomicrons and VLDL by zonal ultracentrifugation in NaBr gradients in the density range 1.0-1.1. Plasma lipoproteins were analyzed for composition of lipids and content of protein, for electrophoretic mobility on paper, and for antigenic determinants by immunoelectrophoresis and immunodiffusion. Flotation constants (S(f)) of the LDL and HDL were calculated from measurements made in the analytical ultracentrifuge. Lipoproteins isolated from plasma by zonal ultracentrifugation were identical by these criteria to lipoproteins isolated by the usual procedure of sequential ultracentrifugation in solvents of increasing density. The procedure of zonal ultracentrifugation is rapid, quantitative, and less laborious than sequential techniques. Lipoproteins isolated by zonal ultracentrifugation are relatively uncontaminated by other proteins and extensive washing is therefore unnecessary. Zonal ultracentrifugation is more than a preparative method for the plasma lipoproteins; it is also an analytical procedure in that a record is obtained of the distribution and quantity of the lipoprotein within the continuous density gradient.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Sequential microultracentrifugation of lipoproteins in 100 microliters of serum

A method is described for sequential separation of high density, very low density, and low density lipoproteins (HDL, VLDL, and LDL, respectively) from 100 microliters of serum, using an air-driven ultracentrifuge (Airfuge, Beckman). Cesium chloride was used for density adjustment. Precision-within-series (coefficient or variation) depended on the cholesterol concentration in the lipoprotein fractions, greater than 1 mmol/l, less than 2.3%. Recovery within-series was nearly 100%. The results (mmol/l) correlate well with those from an electrophoretic-enzymatic procedure (alpha-HDL: 1.49 +/- 0.34 vs. 1.48 +/- 0.33, r = 0.949; pre-beta-VLDL: 0.58 +/- 0.42 vs. 0.59 +/- 0.45, r = 0.975; beta-LDL: 3.11 +/- 0.93 vs. 3.07 +/- 0.88, r = 0.990; n = 48). Recovery of lipoprotein cholesterol from this group was 99.2 +/- 4.2%. A combination of ultracentrifugation with high-performance thin-layer chromatography for determination of lipoprotein-lipid profiles was achieved with recoveries of 98-101%, as evaluated from a group of healthy men (n = 31) and women (n = 38). The entire procedure is, therefore, suitable for compositional studies on lipoproteins from small serum samples. In particular, capillary serum from children of all ages, even from premature neonates, is quite adequate.     

Lipoprotein separation and low density lipoprotein molecular weight determination using high performance gel-filtration chromatography

Plasma lipoproteins were isolated at d less than 1.225 g/ml from nonhuman primates of three species, cynomolgus, rhesus, and African green (vervet) monkeys. Individual lipoprotein classes were separated by high performance gelfiltration chromatography and low density lipoprotein (LDL) molecular weight was determined. A comparison was made using column configurations including TSK 3000 SW, 4000 SW, and 5000 PW columns. Due to its relative simplicity, stability, and economy, a single 5000 PW column was selected for most of the work. The recovery of lipoprotein cholesterol from the column averaged 91 +/- 2.5%. A comparison of the immunologic, chemical, and electrophoretic properties of high density lipoproteins (HDL) and LDL isolated by this technique with those of HDL and LDL isolated by conventional agarose column chromatography indicated that lipoproteins isolated by high performance gel-filtration chromatography were intact and reasonably free of cross contamination. A standard preparation of 125I-labeled LDL was added to the d less than 1.225 g/ml lipoprotein fraction just prior to separation and a relative size index, r1, was determined. When r1 values for a large number of samples were compared with the log of the LDL molecular weight (determined by agarose column chromatography) a linear relationship was found with a correlation coefficient, r = 0.85. The regression equation for this relationship could be used to calculate LDL molecular weights from the r1 value. These values agreed with LDL molecular weight determined by flotation equilibrium analysis in the analytical ultracentrifuge. We conclude that high performance gel-filtration chromatography using the TSK 5000 PW column provides an analytical and preparative technique for simultaneous separation of individual lipoproteins and determination of LDL molecular weight.     

Neutral lipid transfer and lipolysis convert high molecular weight LDL from cholesterol-fed nonhuman primates towards normal: a molecular analysis

In cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet, large, cholesterol ester-rich LDL (Mr greater than 3.5.10(6] are found at the same time that the plasma triacylglycerol levels are low. We studied whether the presence of higher concentrations of triacylglycerol-rich lipoproteins (VLDL) during in vitro incubations would allows depletion from LDL of cholesterol ester and a decreased LDL molecular weight. Three high Mr LDL (Mr = (3.7-4.8).10(6)), rich in cholesterol ester (50 +/- 1.4% by weight), were isolated from three animals by zonal ultracentrifugation, and were then incubated with human VLDL at 37 degrees C for 18 h in lipoprotein-deficient human plasma containing neutral lipid transfer activity. After incubation, modified LDL (M-LDL) was isolated by zonal ultracentrifugation. M-LDL was triacylglycerol-rich (36 +/- 5% by weight) and cholesterol ester-poor (20 +/- 3%), and cholesterol ester had transferred into VLDL. Purified lipoprotein lipase was added to the M-LDL, and triacylglycerol was hydrolyzed. The size of the post-lipolysis M-LDL (Mp-LDL) particles became smaller (mean diameters of 253 A and 228 A for two native LDLs and 215 A and 193 A for Mp-LDL, respectively). Both analytical and zonal ultracentrifugation showed Mp-LDL to be more dense than native LDL. Estimated molecular weights for Mp-LDL were 40%-50% less than that of the original LDL, and fell within the molecular weight range for normal human and monkey LDL. Lipid exchanges, but not apoprotein transfers, were responsible for LDL remodelling, as supported by three separate methods of analysis. Cholesterol ester losses accounted for about two-thirds of the molecular weight decrease. These in vitro results suggest that cholesterol ester enrichment of apoprotein B lipoprotein particles can be reversed by providing adequate levels of VLDL in the presence of neutral lipid transfer processes and lipolytic activity.     

Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent

We have developed a high-performance liquid chromatography (HPLC) method for measurement of cholesterol in the major classes of serum lipoproteins, i.e., HDL, LDL, IDL, VLDL, and chylomicrons. Lipoproteins in serum were separated on a column containing diethylaminoethyl-ligand nonporous polymer-based gel by elution with a step gradient of sodium perchlorate concentration, and detected by post-column reaction with a reagent containing cholesterol esterase and cholesterol oxidase. The within-day assay and between-day assay coefficients of variation for cholesterol concentration in lipoproteins were in the ranges of 0.9-6.4% and 1.1-11.9%, respectively. The correlation coefficients between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol measured by the HPLC method and those estimated by an ultracentrifugation method were 0.892, 0.921, 0.840, 0.930, and 0.873, respectively. Values of remnant-like particle cholesterol measured by an immunoseparation technique (Japan Immunoresearch Laboratories, Japan) were significantly correlated with VLDL and chylomicron cholesterol values measured by the HPLC method (r = 0.883 and r = 0.729, respectively).This rapid and accurate HPLC method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia.     

Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.     

Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.     

Comparison of the lipopretein profiles and the effect of N-phenylpropyl-N-benzyloxy acetamide in primates (38473).

Analytical ultracentrifugation showed Cebus and Rhesus monkeys had two low density components while only one was present in Squirrel monkeys. In untreated or W1372 treated monkeys, neither chylomicrons nor very low density lipoproteins were detected on analytical ultracentrifugation. Chylomicrons were not observed on agarose gel electrophoresis. Ultracentrifugal analysis showed W1372 treatment decreased the amount of LDL in all animals and also the HDL in Cebus monkeys on an atherogenic diet. Both untreated and W1372 treated Cebus monkeys on an atherogenic diet had abnormal amounts of LDL and HDL, while the LDL in treated animals occurred as multiple peaks. This was also evident on agarose gel electrophoresis. Accumulation of lipds in the liver and decrease of serum lipids indicated W1372 prevented release of lipoproteins from the liver.     

Fish oil decreases hepatic cholesteryl ester secretion but not apoB secretion in African green monkeys

Two groups of African green monkeys were fed diets containing 40% of calories as fat with half of the fat calories as either fish oil or lard. The fish oil-fed animals had lower cholesterol concentrations in blood plasma (33%) and low density lipoproteins (LDL) (34%) than did animals fed lard. Size and cholesteryl ester (CE) content of LDL, strong predictors of coronary artery atherosclerosis in monkeys, were significantly less for the fish oil-fed animals although the apoB and LDL particle concentrations in plasma were similar for both diet groups. We hypothesized that decreased hepatic CE secretion led to the smaller size and reduced CE content of LDL in the fish oil-fed animals. Hepatic CE secretion was studied using recirculating perfusion of monkey livers that were infused during perfusion with fatty acids (85% 18:1 and 15% n-3) at a rate of 0.1 mumol/min per g liver. The rate of cholesterol secretion was less (P = 0.055) for the livers of fish oil versus lard-fed animals (3.3 +/- 0.5 vs. 6.0 +/- 1.2 mg/h per 100 g, mean +/- SEM) but the rate of apoB secretion was similar for both groups (0.92 +/- 0.15 vs. 1.01 +/- 0.13 mg/h per 100 g, respectively). The hepatic triglyceride secretion rate was also less (P less than 0.05) for the fish oil-fed animals (8.3 +/- 2.5 vs. 18.3 +/- 4.4 mg/h per 100 g). Liver CE content was lower (P less than 0.006) in fish oil-fed animals (4.1 +/- 0.8 vs. 7.4 +/- 0.7 mg/g) and this was reflected in a lower (P less than 0.04) esterified to total cholesterol ratio of perfusate VLDL (0.21 +/- 0.045 vs. 0.41 +/- 0.06). The hepatic VLDL of animals fed fish oil had 40-50% lower ratios of triglyceride to protein and total cholesterol to protein. From these data we conclude that livers from monkeys fed fish oil secreted similar numbers of VLDL particles as those of lard-fed animals although the hepatic VLDL of fish oil-fed animals were smaller in size and relatively enriched in surface material and depleted of core constituents. Positive correlations between plasma LDL size and both hepatic CE content (r = 0.87) and hepatic VLDL cholesterol secretion rate (r = 0.84) were also found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasma lipoprotein profile and composition in White Carneau and Show Racer breeds of pigeons.

Plasma lipoprotein profile and composition in atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons were investigated while consuming a regular pigeon chow diet free of cholesterol. Plasma was studied by analytical and preparative ultracentrifugation and paper electrophoresis. Lipid composition of each lipoprotein was determined by combined TLC-GLC techniques. The major plasma lipoprotein of both breeds was high density lipoprotein (HDL) with some low density lipoprotein (LDL) and no very low density lipoprotein (VLDL). Cholesterol was mainly found in the HDL in both breeds (71.7%), and no difference was noticed in the total cholesterol content of whole plasma or in various lipoproteins. The LDL fraction in White Carneaux showed a significantly lower (P less than 0.05) percentage of cholesterol esters compared with Show Racers (58.63 +/- 4.9 in White Carneaux vs. 72.12 +/- 2.1 in Show Racers). In LDL, the percentage of the triglyceride concentration in White Carneaux was significantly lower (P less than 0.01) than that of Show Racers while the percentage of protein content in White Carneaux was higher than in Show Racers. No significant differences were observed in fatty acid composition of steryl esters phospholipids, and triglycerides in the lipoprotein fractions of the two breeds. These studies show important differences in the cholesterol esters, protein, and triglyceride content of LDL in the atherosclerosis-susceptible breed of pigeons.     

ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.     

Turnover of 125I-VLDL and 131I-LDL apolipoprotein B in rabbits fed diets containing casein or soy protein

Rabbits fed low-fat, cholesterol-free, semi-purified diets containing casein developed a marked hypercholesterolemia compared to rabbits fed a similar diet containing soy protein (plasma cholesterol 281 +/- 31 vs. 86 +/- 9 mg/dl; P less than 0.05). Turnover studies (three per dietary group) were carried out in which homologous 125I-labeled VLDL and 131I-labeled LDL were injected simultaneously into casein- (n = 8) or soy protein- (n = 9) fed rabbits. ApoB-specific activities were determined in VLDL, IDL and LDL isolated from the pooled plasma of two or three rabbits per dietary group. The production rate of VLDL apoB (1.20 +/- 0.3 vs. 1.09 +/- 0.1 mg/h per kg) was similar for the two dietary groups. The fractional catabolic rate of VLDL apoB was lower for the casein group (0.15 +/- 0.03 vs. 0.23 +/- 0.01.h-1; 0.05 less than P less than 0.10). Although the pool size of VLDL apoB was higher in the casein group (8 +/- 2 vs. 5 +/- 0.3 mg/kg), this value did not reach statistical significance. For LDL apoB, the increased pool size in casein-fed rabbits (30 +/- 5 vs. 5 +/- 1 mg/kg; P less than 0.01) was associated with a decreased fractional catabolic rate (0.03 +/- 0.005 vs. 0.08 +/- 0.008.h-1; P less than 0.01) and a 2-fold increase in the production rate of LDL apoB (1 +/- 0.3 vs. 0.4 +/- 0.06 mg/kg per h; 0.05 less than P less than 0.10) compared to rabbits fed soy protein. Analysis of precursor-product relationships between the various lipoprotein fractions showed that casein-fed rabbits synthesized a higher proportion of LDL apoB (95% +/- 2 vs. 67% +/- 2; P less than 0.001) independent of VLDL catabolism. These results support the concept that the hypercholesterolemia in casein-fed rabbits is associated with impaired LDL removal consistent with a down-regulation of LDL receptors. These changes do not occur when the casein is replaced by soy protein.     

A new micromethod for routine measurement of serum LDL-apolipoprotein B

A new method for low density lipoprotein (LDL) (d 1.019-1.063 g/ml)-apolipoprotein B (apoB) determination has been developed, based on the fact that very low density and intermediate density lipoproteins (VLDL and IDL) contain apolipoprotein C-I (apoC-I), whereas this apolipoprotein is apparently absent in LDL. VLDL and IDL were quantitatively precipitated with a monospecific anti-apoC-I antibody whereafter LDL-apoB in the supernatant was quantitated by Laurell rocket electrophoresis. Over a wide range of cholesterol and triglyceride values there was a linear correlation with LDL-apoB values measured after ultracentrifugation. The method would be useful for routine measurements, especially in children, since only 25 microliter of serum is required, and for making the diagnosis of hyperapobetalipoproteinemia, which at present is complicated.     

A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass

Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoprotein structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradients were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of LDL subclass pattern.     

Heterogeneity of serum lipoproteins during the fetal and neonatal development of the pig

Serum lipoproteins from fetal, neonatal and adult pigs were characterized with the use of lipid analysis, polyacrylamide gradient gel electrophoresis, two-dimensional immunoelectrophoresis and zonal ultracentrifugation. Almost all serum cholesterol was found in LDL during the early stages of fetal development, while low but increasing levels appeared in the fetal pig HDL by the end of the gestation period. In the fetal pig, most of the serum triglycerides could be found in the HDL fraction. After the start of suckling, the levels of serum triglycerides and cholesterol increased. Most of these exogenous lipids were found in the chylomicrons + VLDL + LDL fraction of the newborn pig serum. The molecular weights of the native serum lipoproteins were calculated as being 2.0-2.4 X 10(5) daltons for newborn pig HDL and 1.4-1.7 X 10(6) daltons for newborn pig LDL. Minor changes in the molecular weight distributions were detected within these ranges for both HDL and LDL during fetal and neonatal development of the pig. Zonal ultracentrifugation of neonatal pig serum partly separated the LDL into three subfractions, whereas neonatal HDL appeared as one broad fraction.     

Physical and kinetic properties of homogenous bovine lens aldose reductase.

Aldose reductase from calf lens was purified 15,000-fold. The homogeneity of the final preparation was demonstrated by molecular sieve chromatography, analytical ultracentrifugation, sodium dodecyl sulfate gel electrophoresis, Ouchterlony immunodiffusion, and polyacrylamide gel electrophoresis at three pH values. The monomeric nature of the enzyme is suggested by the molecular weight of 37,000 from both molecular sieve chromatography and sodium dodecyl sulfate-gel electrophoresis with beta-mercaptoethanol. This closely corresponds with a molecular weight of 40,400 estimated by using calculate physical constants in the Svedberg equation. The S20,w was 3.6 to 3.7 as determined from ultracentrifuge and sucrose density gradient data. The Stokes radius was found to be 2.5 +/- 0.2 nm and 2.75 +/- 0.15 nm by two different methods. The diffusion constant D20,w is (7.8 +/- 10(-7) +/- 0.45 X 10(-7) cm2/s). The molecule is nearly spherical as indicated by a frictional ratio f/fo = 1.14. The alpha-helical content was estimated from circular dichroism data to be 5% and did not change in the presence of added substrates, products, and some enzyme inhibitors. Homotropic cooperative effects were observed as shown by the concave downward curvature of the reciprocal plots.     

Altered lipoprotein subclass distribution and PAF-AH activity in subjects with generalized aggressive periodontitis

In this study, we examined whether the documented increase of plasma triglycerides in patients with generalized aggressive periodontitis (GAgP) is associated with changes in lipoprotein subclass distribution and/or LDL-associated platelet-activating factor acetylhydrolase (PAF-AH) activity. Lipoprotein subclasses were analyzed in whole plasma samples using nuclear magnetic resonance methods. Compared with subjects without periodontitis (NP subjects; n = 12), GAgP subjects (n = 12) had higher plasma levels of large, medium, and small VLDL (35.0 +/- 6.7 vs. 63.1 +/- 9.6 nmol/l; P = 0.025), higher levels of intermediate density lipoprotein (24.8 +/- 11.6 vs. 87.2 +/- 16.6 nmol/l; P = 0.006), lower levels of large LDL (448.3 +/- 48.5 vs. 315.8 +/- 59.4 nmol/l; P = 0.098), and higher levels of small LDL (488.2 +/- 104.2 vs. 946.7 +/- 151.6 nmol/l; P = 0.021). The average size of LDL from NP and GAgP subjects was 21.4 +/- 0.2 and 20.6 +/- 0.3 nm, respectively (P = 0.031). Compared with NP subjects, GAgP subjects had a greater number of circulating LDL particles (961.3 +/- 105.3 vs. 1,349.0 +/- 133.2 nmol/l; P = 0.032). Differences in the plasma levels of large, medium, and small HDL were not statistically significant. NP and GAgP subjects had similar plasma levels of total LDL-associated PAF-AH activity; however, LDL of GAgP subjects contained less PAF-AH activity per microgram of LDL protein (1,458.0 +/- 171.0 and 865.2 +/- 134 pmol/min/microg; P = 0.014). These results indicate that, in general, GAgP subjects have a more atherogenic lipoprotein profile and lower LDL-associated PAF-AH activity than NP subjects. These differences may help explain the increased risk of GAgP subjects for cardiovascular disease.