Waldo R. Fisher

Heterogeneity of plasma low density lipoproteins manifestations of the physiologic phenomenon in man

Abstract Plasma low density lipoproteins (LDL) consists of a family of lipoproteins, having densities between 1.006 and 1.063 gm/ml, which are either physically monodisperse or polydisperse in different individuals. In monodisperse LDL most of the mass of lipoprotein is found within a narrow region of this density range, and the preponderance of the lipoprotein is similar in molecular weight. By contrast, polydisperse LDL is heterogeneous in molecular size, and the lipoprotein mass is distributed over the LDL density range. An examination of LDL heterogeneity in 187 subjects reveals that normolipemic and hypercholesterolemic subjects have predominantly monodisperse LDL. The molecular weight range of such LDL is 2.1–3.9 × 106, and for a given subject the molecular weight varies minimally with different metabolic pertubations. The major determinant of the molecular weight of monodisperse LDL appears to be genetic, modified by metabolic influences. In contrast, about 80% of individuals with hypertriglyceridemia have polydisperse LDL, and hypertriglyceridemic subjects with diabetes have a similar incidence of LDL polydispersidy. LDL heterogeneity increases with increasing VLDL concentration. Polydisperse LDL have an increased apoprotein content compared to monodisperse LDL and compositional analysis reveals an increased protein to lipid ratio. Current knowledge of the biosynthesis of LDL affords an incomplete undrstanding of why LDL is monodisperse in some individuals and polydisperse in others. The relationship between LDL dispersion and atherosclerosis has been examined in 105 subjects. Though the epidemiologic data are tentative, they suggest an increased association of polydisperse LDL with atherosclerosis in hypertriglyceridemic diabetics though not in other individuals.

ApoB metabolism in familial hypercholesterolemia. Inconsistencies with the LDL receptor paradigm.

The biology of the low-density lipoprotein (LDL) receptor has been examined in detail, and a paradigm for LDL metabolism has evolved from comparative studies of cholesterol metabolism in a variety of cells cultured from normal individuals and subjects with familial hypercholesterolemia (FH). Cultured cells from patients with homozygous FH lack a functional LDL receptor and show diminished LDL clearance, induction of the enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, increased cholesterol synthesis, decreased cholesterol ester production, and depleted cholesterol ester stores. The observed decrease in the fractional catabolic rate (FCR) of LDL is attributed to the mutated LDL receptor gene. However, in the experimental animal model of this disease, the Watanabe heritable hyperlipidemic (WHHL) rabbit, cholesterol ester stores are increased, while hepatic cholesterol synthesis is decreased. Furthermore, in humans HMG-CoA reductase is suppressed, and the LDL apolipoprotein (apo) B production rate is increased in patients with FH. These findings raise questions about the adequacy of the paradigm in understanding hepatic cholesterol metabolism in vivo. In humans, apoB metabolism is believed to be principally determined by the liver, where apoB is both synthesized and catabolized. Assuming the neutral lipid content of the liver is the major determinant of apoB metabolism, we postulated that the changes in apoB metabolism in FH are predictable when based on the assumption of an increase in hepatic cholesterol and cholesterol ester content, as observed both in the WHHL rabbit and in humans. We examined this hypothesis in vivo in patients with heterozygous FH by using tracer kinetic methodology and have used similar data from normal and hypertriglyceridemic (HTG) subjects as controls. Whereas normal and HTG subjects secrete apoB primarily as large, triglyceride-enriched very-low-density lipoprotein (VLDL), heterozygous FH patients have an absolute decrease in apoB production and secrete almost 40% of apoB as smaller intermediate-density lipoprotein (IDL)/LDL. In normal humans, about half of secreted apoB is catabolized rather than being converted to LDL. In HTG subjects two thirds of apoB follows this same route, by which VLDL remnants remaining after triglyceride hydrolysis are largely returned to the liver. In contrast, in FH subjects secreted apoB is fully converted to LDL. Thus, although total apoB secretion is reduced in FH subjects, total LDL production is greater than in either normal or HTG subjects. Under basal conditions the elevated LDL in heterozygous FH is due to both decreased LDL receptor-mediated catabolism and increased LDL production. However, the number of LDL receptors actually expressed is suppressed below the number of potentially functional receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

[11] Isolation and characterization of apolipoprotein B-100

Publisher Summary Two physically separable macromolecules carry the designation apolipoproteinB. The long recognized form of apoB, large apoB or B-100, is the predominant protein in LDL and the major apoB protein found in human VLDL. This chapter describes the isolation and characterization of apolipoprotein B-100. ApoB-100 is generally an abundant apolipoprotein in plasma, and constitutes over 95% of the protein mass of low-density lipoprotein (LDL) and approximately one-third of the protein mass of very low-density lipoprotein. It is readily isolated from LDL. ApoB may be modified chemically with lysines and arginines, which changes the biological properties of the apolipoprotein. The chapter suggests that the amino acid analysis of apoB is not particularly useful in characterizing a preparation, and it has been shown that up to 30% of the protein may be removed enzymatically with little change in the amino acid composition of the remaining apoB. A minimal criterion for characterizing an apoB preparation is demonstration of a single band on a 4 or 5% SDS-polyacrylamide gel, and a second criterion is the measurement of the sedimentation coefficient.

Macromolecular dispersion of human plasma low-density lipoproteins in hyperlipoproteinemia☆

Abstract Low-density lipoprotein (LDL) has previously been reported to exist in either a polydisperse or a monodisperse state. Using the techniques of analytical velocity sedimentation and/or equilibrium density-gradient ultracentrifugation, the macromolecular dispersion of LDL has been investigated in 139 subjects classified by their lipoprotein phenotypes as follows: 63 normal, 25 type IIA, 6 type IIB, and 45 type IV. LDL polydispersion was found in 78% of subjects with hypertriglyceridemia (type IIB or IV phenotypes), while only 9% of normotriglyceridemic subjects had polydisperse LDL. A study of LDL dispersion in two families, one with hyperpreβ-lipoproteinemia and one with combined hyperlipoproteinemia, also demonstrated the frequent association of LDL polydispersity with increased plasma very low density lipoprotein (VLDL) concentrations. LDL polydispersion results from the presence of higher molecular weight, lipid-enriched lipoproteins of the LDL class. Among hyperlipoproteinemic subjects with a type IV phenotype and with polydisperse LDL, the concentrations of these higher molecular weight subspecies of LDL appear to increase with severity of the hyperlipemia. In 6 subjects, reduction of VLDL concentration resulted in a decrease in the concentration of the higher molecular weight LDL; however, LDL remained polydisperse. By contrast, approximately one-third of subjects with hypertriglyceridemia were observed to have monodisperse LDL, even in the presence of high VLDL concentrations. This observation raises the possibility of two separate populations of subjects with hypertriglyceridemia arising from increased VLDL concentration.

The absence of a role for the carbohydrate moiety in the binding of apolipoprotein B to the low density lipoprotein receptor

The binding of low density lipoprotein (LDL) to fibroblasts occurs through apolipoprotein B, a glycoprotein. The role of the carbohydrate in binding was assessed in two ways: (1) LDL, freed of sialic acid and most of the glucosamine and hexoses by digestion with a mixture of glycosidases, bound to fibroblasts as does native LDL. (2) The glycopeptides liberated from apoprotein B by trypsin and pronase failed to inhibit LDL binding to fibroblasts. Apparently the carbohydrate moiety of LDL does not interact with the plasma membrane receptor.

Lipodystrophic diabetes mellitus. Investigations of lipoprotein metabolism and the effects of omega-3 fatty acid administration in two patients☆

We investigated the metabolic effects of omega-6 (safflower oil) and omega-3 (fish oil) fatty acid-enriched diets (65% carbohydrate, 20% fat) in two patients with a syndrome of diabetes mellitus, lipodystrophy, acanthosis nigricans, chylomicronemia, and abdominal pain. 3H-glycerol was used to evaluate triglyceride-rich lipoprotein-triglyceride (TRLP-TG) metabolism, and changes in glucose and insulin dynamics were also studied. On the omega-6 diet, both subjects demonstrated four- to five-times normal rates of TRLP-TG production and glycerol biosynthesis, and striking decrements in the fractional catabolic rate (FCR) for TRLP-TG and TRLP-particles. Both subjects had elevations in nonesterified fatty acid (NEFA) concentrations. In one patient, the omega-3 diet markedly decreased serum triglycerides and newly synthesized triglyceride glycerol production, in association with a fall in NEFA. In both subjects, plasma glycerol reutilization for triglyceride synthesis, normal on the omega-6 diet, was abolished on the omega-3 regimen. Plasma postheparin lipolytic activity was normal on both diets. On the omega-3 diet, xanthomas and hepatomegaly decreased and, in the patient who had no reduction in serum triglycerides, pancreatitis attacks virtually ceased. Mean 24-hour serum glucose levels were higher, and both basal and peak C-peptide responses to a carbohydrate meal were blunted on the omega-3 diet. One patient became ketonuric. We conclude the cause of hypertriglyceridemia in these patients was due to increased lipid synthesis and hypothesize that this is secondary to high plasma concentrations of NEFA. In addition, an omega-3 diet in these subjects inhibited insulin secretion and worsened glucose tolerance.

Medical Student Research: A Program of Self-Education.

Student participation in research has traditionally been part of the academic program in American medical schools. A survey of student research in 115 U.S. medical schools showed that 74 percent had M.D./Ph.D. programs but that only about 0.6 percent of the students participated in them. Eighty percent had research programs not directed toward a Ph.D.; and although these involved only 6 percent of the students, the enthusiasm of the students for their research experience was very high. The University of Florida has long fostered medical student research. A survey of 1969--1978 participants in the Florida programs yielded a 74 percent endorsement of research as an important, positive educational experience, and nearly all agreed that a research option should be part of the medical school curriculum. Since research provides the students with an intellectually challenging, self-learning experience that they endorse enthusiastically and since there is small student participation, it seems timely to reassess the role of student research in the medical school curriculum.

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.

Structural studies of apolipoprotein b physical properties of the protein in guanidine hydrochloride

Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (Mz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540,000 +/- 110,000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25 degrees C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640,000 and 250,000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.

A comparative study of enzymatic digestion profiles of apolipoprotein B from four human subjects.

A methodological approach for comparative structural study of apolipoprotein B has been developed. Low-density lipoproteins from four human subjects were digested in three separate enzyme systems, utilizing trypsin, chymotrypsin and Staphylococcus aureus protease V8, each in the presence of 1% sodium dodecyl sulfate. The peptides were separated by electrophoresis on polyacrylamide gels in SDS; the stained gels were scanned spectrophotometrically to produce characteristic profiles. Comparison of the profiles revealed good reproducibility and a high degree of similarity among the different subjects. Of the four subjects studied, one subject had one apparent difference in the tryptic digest profile and also in the S. aureus protease V8 digest profile. The structural significance of these variations can be evaluated only after a larger number of subjects, including those presented here, have been examined; this study is now in preparation.