Steven T. Kunitake

Apolipoprotein L, a New Human High Density Lipoprotein Apolipoprotein Expressed by the Pancreas IDENTIFICATION, CLONING, CHARACTERIZATION, AND PLASMA DISTRIBUTION OF APOLIPOPROTEIN L

In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas. The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction. The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids. No significant homology was found with known sequences. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa. Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa. Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2–17 and 10.4–12.2 nm. Moreover, Lp(L) exhibited both pre-β and α electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles.

Complementarity of Colestipol, Niacin, and Lovastatin in Treatment of Severe Familial Hypercholesterolemia

OBJECTIVE To compare the effectiveness of the ternary-drug combination of colestipol, niacin, and lovastatin with binary combinations of those drugs in treating patients with familial hypercholesterolemia. DESIGN An open sequential study of serum lipoprotein responses in patients receiving diet alone (mean duration, 4 months); colestipol and niacin with diet (mean duration, 9 months); and colestipol, niacin, and lovastatin with diet (mean duration, 15 months). SETTING Metabolic ward and lipid clinic of a university medical center. PATIENTS Twenty-two patients with clinical characteristics of familial hypercholesterolemia (low-density-lipoprotein cholesterol, greater than 8.48 mmol/L; 21 of 22 with tendon xanthomas). INTERVENTIONS Diet: less than 200 mg/d of cholesterol and less than 8% of total calories from saturated fat; colestipol, 30 g/d; lovastatin, 40 to 60 mg/d; and niacin, 1.5 to 7.5 g/d. MEASUREMENTS AND MAIN RESULTS Mean total serum cholesterol and low-density-lipoprotein cholesterol levels of 4.86 +/- 0.62 mmol/L (188 +/- 24 mg/dL SD) and 2.89 +/- 0.54 mmol/L (112 +/- 21 mg/dL SD), respectively, were significantly lower during ternary-drug treatment than during colestipol-niacin treatment (p less than 0.003) or during treatment in which other possible binary combinations were given. The cholesterol content of very low-density-lipoproteins was lower and high-density-lipoprotein cholesterol levels higher during this phase than during the colestipol-niacin phase. CONCLUSIONS Colestipol, lovastatin, and niacin are mutually complementary in treating hypercholesterolemia. This regimen produces reductions in serum cholesterol levels similar to those associated with regression of atheromatous plaques in animal studies.

Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.

Heterogeneity of high-density lipoproteins and apolipoprotein A-I as related to quantification of apolipoprotein A-I.

Publisher Summary Apolipoprotein (apo) A-I is the major protein constituent of the human plasma high-density lipoproteins (HDL). It is present on the bulk of the HDL particles, isolated from the fasting plasma of normolipidemic individuals. Accurate measurement of apoA-I is central to the study of HDL. Plasma levels of apoA-I correlate well with plasma HDL cholesterol levels and it has been suggested that apoA-I may actually be a more accurate predictor of cardiovascular risk than HDL cholesterol. In addition, HDL complexes are heterogeneous, with respect to protein constituents and, therefore, an accurate determination of the apoA-I will aid in the elucidation of the apolipoprotein stoichiometry of the various complexes and the distribution of apoA-I among them. Direct quantitation of apoA-I, by immunoassay, is complicated, by variability in the epitopes of apoA-I. This variability may arise either from the structural differences in the apoA-I protein itself or from the conformational differences, owing to the association of apoA-I with distinct HDL species.

Marine Mammals: Animal Models for Studying the Digestion and Transport of Dietary Fats Enriched in w-3 Fatty Acids. Positional Analyses of Milk Fat Triacylglycerol Molecules

In the depot fat of both marine and terrestrial animals, the relative percent distribution of the major fatty acids at the three positions of the glycerol backbone are not the same. Interestingly, studies have revealed that the polyenoic omega-3 fatty acids in marine mammalian blubber and fish oils are distributed differently. In marine mammalian blubber, the polyenoic omega-3 fatty acids are located on the outer positions of the molecule, i.e. the 1- and 3-positions. In fish oils, the polyenoic omega-3 fatty acids are located almost exclusively in the 2-position.