Donald L. Puppione

[6] Sequential flotation ultracentrifugation

Publisher Summary Plasma lipoproteins have lower hydrated densities relative to the other plasma proteins; therefore sequential flotation ultracentrifugation has been the principal method used for their isolation and classification. This chapter describes the sequential flotation ultracentrifugation method and some important features for the proper use of sequential flotation ultracentrifugation, such as it is important to calculate the effect of differences in path length or it is virtually impossible to reduce the level of contaminating proteins, such as albumin, below detection limits with a single spin. The ability to process simultaneously large volumes or many small samples is one of the principal advantages of sequential flotation ultracentrifugation. However, the technique suffers from certain limitations, such as during the course of long centrifugation, extensive lipid peroxidation can occur. This problem may be minimized by the inclusion of appropriate chelating agents and antioxidants. Full fractionation and purification by ultracentrifugal methods are both lengthy and costly. Also sequential flotation ultracentrifugation separates lipoproteins according to density only, and the investigator must be aware that particles differing in stage of metabolism, composition, charge, and size may be combined in any of the fractions obtained with this method.

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci.

We have tested for evidence of linkage between the genetic loci determining concentrations and composition of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes participating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple sequence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determining plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) locus on chromosome 6 (P = 0.02). Thus, these data indicate the simultaneous involvement of at least two different genetic loci in the determination of the levels of HDL and its associated lipoproteins.

Serum lipoproteins in the spiny lobster, Panulirus interruptus

1. Most of the lipids in the hemolymph of the spiny lobster, Panulirus interruptus, were associated with a high density lipoprotein (HDL3). The lipid of this lipoprotein was composed of phospholipid (88%), sterol (4%) and triglyceride (3%). 2. In animals fed 14C-labeled triglyceride radioactivity was not seen in the serum until 12 hr after feeding. Most of this serum radioactivity was associated with phosphatidyl choline. 3. Electron micrographs showed that negatively stained high density lipoproteins of the lobster had a polymorphic appearance.

Physicochemical study of rock crab lipoproteins

Physicochemical studies have been carried out on the hemolymph and egg lipoproteins of the rock crab (Cancer antennarius). Analytical ultracentrifugal analyses of vitellogenic female HDL3 revealed the presence of two types of lipoproteins. The first with a sedimentation rate of 5.35 S was comparable to lipoproteins in male and non-vitellogenic female hemolymph. The second with a sedimentation rate of 10.74 S was comparable to the major lipoprotein of egg yolk. A similar comparison could be made following electrophoretic analyses in native polyacrylamide gels. Electrophoresis in SDS-polyacrylamide gels revealed three major apolipoproteins common to egg and vitellogenic HDL3. A fourth apolipoprotein was found in both male and female HDL3. In contrast to mammalian HDL, none of these crustacean apolipoproteins had a molecular weight less than 82 000. One of these apolipoproteins appears to be comparable physicochemically to the enteric form of apolipoprotein B in mammals.

Homocysteinylated Albumin Promotes Increased Monocyte-Endothelial Cell Adhesion and Up-Regulation of MCP1, Hsp60 and ADAM17

Rationale The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties. Objective Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels. Methods and Results Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2. Conclusions Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.

A microprecipitation technique suitable for measuring α-lipoprotein cholesterol

A semi-automated method has been developed for determining α-lipoprotein cholesterol values. Precipitation of apolipoprotein B containing lipoproteins takes place in wells of microtiter plates after 100 μL of serum are mixed with 20 μL of a heparin/MnCl2 solution. A Beckman (Fullerton, CA) Biomek 1000 work station is used to transfer sera, supernatants and reagents between tubes and microtiter plates. Supernatant cholesterol is determined enzymatically, and absorbances are read at 490 nm using a Molecular Devices Corporation (Palo Alto, CA) plate reader. Values obtained on both fresh and frozen serum samples agreed with corresponding data obtained at the Centers for Disease Control (CDC; Atlanta, GA). For the fresh samples, the average bias was 2.87%. The within-run coefficients of variations were between 2.2 and 0.6% for the data obtained on CDC frozen control pools. The results indicate that the semi-automated method is suitable for obtaining accurate and precise data for α-lipoprotein cholesterol. The method lends itself to the analysis of large numbers of samples and is particularly suited for the study of lipoproteins of small mammals.

Relationships among serum lipids, milk production and physiological status in dairy cows

Abstract 1. 1. Serum concentrations of cholesterol, triglyceride and alpha lipoprotein cholesterol were determined in nine Holstein cows during different stages of their gestation and lactation cycles. 2. 2. Serum cholesterol concentrations and daily milk yields were significantly correlated during early and late lactation. The correlations were not significant over the entire lactation. 3. 3. Maximum values for serum cholesterol were observed at mid lactation and minimum at or near parturition. Serum triglyceride concentrations ranged from 4 to 33 mg/dl. Although prepartum levels for triglyceride tended to be higher than postpartum concentrations, no correlation with milk yield was noted. 4. 4. These data are compared with lipid changes which take place in other mammals during lactation.

Serum lipoproteins in two species of phocids (Phoca vitulina and Mirounga angustirostris) during alimentary lipemia

Abstract 1. 1. Serum lipoprotein distributions of harbor seals ( Phoca vitulina ) and elephant seals ( Mirounga angustirostris ) during alimentary lipemia were measured by analytical ultracentrifugation. 2. 2. Lipid compositions and concentrations of the total VLDL, LDL, and HDL classes were also determined. During alimentary lipemia increased serum triglycerides were associated with concentration changes in the total VLDL class. A postprandial increase in HDL phospholipid was observed in one animal. 3. 3. Comparison of lipid concentration of the total VLDL class with the S° ∝ 20–400 data indicates that chylomicra are the principal triglyceride carriers during alimentary lipemia and that there are only slight changes among the S ∝ 20–400 lipoproteins. 4. 4. Electrophoretic analyses were carried out on sera obtained from elephant seals. Chylomicron and pre-beta bands were detected, but there was no discernible beta band. Consistent with the latter finding no S ° ∝ 0–20 lipoproteins were observed in the analytical ultracentrifuge. 5. 5. These data are discussed in terms of the clearance of chylomicron remnants.

Microsequencing of bovine cerebrospinal fluid apolipoproteins: Identification of bovine apolipoprotein E

In studies of bovine plasma lipoproteins, apolipoprotein E (apoE) was not found associated with α-lipoproteins isolated over a broad range of densities. However, studies of cerebrospinal fluid (CSF) lipoproteins from other mammals have shown that apoE is a major apolipoprotein associated with high density lipoprotein, a fact that prompted us to determine if this were also the case in bovine CSF. CSF samples were obtained from animals with a surgically implanted catheter. Most analyzed samples were obtained from cows at various stages of the postpartum period; however, a few samples also were obtained at term or during pregnancy. Analyses of isolated ultracentrifugal fractions by polyacrylamide gel electrophoresis revealed the presence of two apo, with the expected molecular weights for apoE and apoA-I. By using both matrix-assisted laser desorption mass spectrometry and microsequencing techniques, we demonstrated that these apo are indeed apoE and apoA-I.

Higher primates, but not New World monkeys, have a duplicate set of enhancers flanking their apoC-I genes.

Previous studies have demonstrated that the apoC-I gene and its pseudogene on human chromosome 19 are flanked by a duplicate set of enhancers. Multienhancers, ME.1 and ME.2, are located upstream from the genes and the hepatic control region enhancers, HCR.1 and HCR.2, are located downstream. The duplication of the enhancers has been thought to have occurred when the apoC-I gene was duplicated during primate evolution. Currently, the only primate data are for the human enhancers. Examining the genome of other primates (great and lesser apes, Old and New World monkeys), it was possible to locate the duplicate set of enhancers in apes and Old World monkeys. However, only a single set was found in New World monkeys. These observations provide additional evidence that the apoC-I gene and the flanking enhancers underwent duplication after the divergence of Old and New World monkeys.