Howard A. Eder

THE DISTRIBUTION AND CHEMICAL COMPOSITION OF ULTRACENTRIFUGALLY SEPARATED LIPOPROTEINS IN HUMAN SERUM

In the past few years several methods have been developed for the analysis of serum lipoproteins. Lindgren, Elliott, and Gofman (1) have utilized the relatively low density of the lipoproteins to separate them from the other serum proteins by ultracentrifugal flotation. Quantitation was subsequently performed by refractometric methods in the analytical ultracentrifuge. Separations of lipoproteins have also been made by Cohn fractionation in cold ethanol, and the quantities of lipoprotein have been estimated from the lipid. content of the fractions (2, 3). Widely used at the present time is the method of zone electrophoresis with quantitation either by staining (4) or by chemical analysis of eluates from the support

Plasma lipids and lipoproteins and the incidence of cardiovascular disease in the very elderly. The Bronx Aging Study.

The Bronx Aging Study is a 10-year prospective investigation of very elderly volunteers (mean age at study entry, 79 years; range, 75-85 years) designed to assess risk factors for dementia and coronary and cerebrovascular (stroke) diseases. Entry criteria included the absence of terminal illness and dementia. All subjects (n = 350) included in this report had at least two lipid and lipoprotein determinations. Overall, more than one third of subjects showed at least a 10% change in lipid and lipoprotein levels between the initial and final measurements. Moreover, mean levels for women were consistently different than those for men, and because of this finding subjects were classified into potential-risk categories based on the changes observed by using their sex-specific lipid and lipoprotein distributions. The incidences of cardiovascular disease, dementia, and death were compared between risk groups. Proportional-hazards analysis showed that in men a consistently low high density lipoprotein cholesterol level (less than or equal to 30 mg/dl) was independently associated with the development of myocardial infarction (p = 0.006), cardiovascular disease (p = 0.002), or death (p = 0.002). For women, however, a consistently elevated low density lipoprotein cholesterol level (greater than or equal to 171 mg/dl) was associated with myocardial infarction (p = 0.032). Thus, low high density lipoprotein cholesterol remains a powerful predictor of coronary heart disease risk for men even into old age, while elevated low density lipoprotein cholesterol continues to play a role in the development of myocardial infarction in women. The findings suggest that an unfavorable lipoprotein profile increases the risk of cardiovascular morbidity and mortality even at advanced ages for both men and women.

Polypeptide composition of rat high density lipoprotein: Characterization by SDS-gel electrophoresis

Summary Electrophoresis on sodium dodecylsulfate-containing polyacrylamide gels of high density lipoprotein obtained from plasma of sucrose-fed rats reveals a complex pattern of three major bands and several minor, low molecular weight bands. The component present in the greatest amount is a 27,000 molecular weight protein which has aspartic acid or asparagine as N-terminus and alanine as C-terminus, and which is considered comparable to the A-I protein of human high density lipoprotein. The other major rat polypeptides have molecular weights of 46,000 (A-IV) and 35,000 (“arginine-rich”). The “arginine-rich” protein may be identical to a major polypeptide component of rat very low density lipoprotein.

Serum lipoproteins and apolipoproteins in rats with streptozotocin-induced diabetes.

The lipoproteins of rats fed a high sucrose diet and made diabetic by administration of 45 mg/kg of streptozotocin were studied. All lipoprotein classes were found to be present in increased concentrations. The apolipoprotein composition of the various lipoprotein fractions was studied by polyacrylamide-gel electrophoresis in the presence of 8 M urea, isoelectric focusing in the presence of 8 M urea, and sodium dodecyl sulfate gel electrophoresis in polyacrylamide gels. In the very low density lipoproteins (VLDL) of diabetic rats, there was a marked alteration in the relative amounts of C proteins by polyacrylamide-gel electrophoresis, and this was found by isoelectric focusing to be primarily a relative increase in C-III-3 apoprotein and a decrease in C-III-O. In addition, in the diabetic rats, the VLDL contained a protein of mol wt 46,000, the A-IV protein, which normally is only present in the high density lipoproteins. In the high density lipoproteins, (HDL) the same alterations in pattern of the C proteins seen in the VLDL were present. Furthermore, the arginine-rich and A-IV protein normally present in HDL could not be detected in the HDL, although the other apolipoproteins are present. Apolipoprotein concentrations were determined by quantitative immunoelectrophoresis. It was found that in the diabetic rats there was an increase in the total amount of apo-B in the plasma, with the increment divided proportionately between the VLDL and the low density lipoprotein (LDL). The total apo-C concentration of plasma increased minimally. The A-IV concentration of plasma increased by 27%; it decreased markedly in the HDL, but appeared in increased amounts in both VLDL and in the d greater than 1.21 fraction. The arginine-rich protein decreased by 63% in the plasma and decreased significantly in the HDL, but increased in VLDL, LDL, and in the d greater than 1.21 fraction. These alterations in apolipoprotein patterns in diabetic animals suggest that the apolipoproteins may play an important role in determining the concentration of various lipoprotein fractions, or may be the result of altered metabolism of the lipoproteins. These lipoproteins with altered apolipoprotein composition may have important biologic differences from normal lipoporteins. Nevertheless, the HDL, despite the fact that it is deficient in some of its major constituents, was unchanged in its cholesterol content.

DIETARY TREATMENT OF HYPERTENSION. CLINICAL AND METABOLIC STUDIES OF PATIENTS ON THE RICE-FRUIT DIET

This report is based on a study of six patients who were treated for hypertension by means of the rice-fruit diet of Kempner (1). During the six months of study with the patients in continuous residence on a metabolic ward observations were made to follow the evolution of both clinical and metabolic changes. The purpose of this study was the analysis of certain aspects of the necessity and the sufficiency of the rice-fruit program as applied to selected patients in a controlled environment. To this end evidence will be presented to demonstrate that objective clinical improvement actually occurred in these patients while they were under detailed metabolic observation. Whether the high incidence of clinical improvement observed was due to the selection of relatively young and uncomplicated hypertensive subjects or due to the detailed supervision of dietary intake or even whether the sheltered environment of a metabolic ward may have contributed to the favorable effects was not investigated. These considerations, although pertinent to the general problem of dietary treatment (2), are not directly relevant to the present issue of association between clinical and metabolic change.

Hyperlipoproteinemia in Streptozotocin-treated Rats

In order to study hyperlipidemia in diabetes mellitus, rats were made diabetic by administration of streptozotocin and the optimal conditions for production of severe and persistent hyperlipoprotenemia determined. Two groups of rats were compared: rats fed sucrose-rich diets and rats fed laboratory chow. The optimal dose of streptozotocin was 45 mg/kg body weight for the sucrose-fed rats. With this dose, plasma glucose reached a maximum of over 600 mg./100 ml., and plasma insulin was reduced by 60 per cent. Plasma triglycerides rose in the sucrose-fed rats to over 1,000 mg/100 ml. two days after the streptozotocin was given and then decreased to over 770 mg./100 ml. 12 days after treatment and then to 585 mg./100 ml. 10 weeks after induction of diabetes. With this dose, ketonuria did not occur nor did any of the animals die, as occurred with higher doses. In the chow-fed rats, plasma triglyceride levels were elevated with the induction of diabetes to levels of approximately 300 mg./100 ml. The concentration of all the plasma lipoproteins increased with the induction of diabetes. The concentration of very-low density lipoprotein (VLDL) protein in the sucrose-fed diabetic increased fivefold, the low-density lipoprotein (LDL) protein increased, and especially striking was the increase in high-density lipoprotein (HDL) protein concentration, which became more pronounced with the duration of the diabetes. The diabetes produced by streptozotocin administration to sucrose-fed rats, thus, provides a useful model for the study of the hyperlipoproteinemia of diabetes.

Extrahepatic synthesis of lipoproteins of plasma and chyle: role of the intestine.

Several groups of investigators (1-3) have shown that the protein portion of the plasma lipo-proteins of the rat can be synthesized by the liver. The present studies were undertaken to determine whether any lipoprotein synthesis can occur in the absence of the liver. Previous work by Bragdon (4) and by Rodbell, Fredrickson, and Ono (5) has suggested intestinal synthesis of lipoproteins. Since their studies were carried out in intact animals, however, the liver could not be excluded as a source of these proteins. We have therefore measured the synthesis of the protein component of the lipoproteins of chyle and plasma in hepatectomized dogs. Methods Male mongrel dogs weighing 12 to 18.5 kg were fasted for 18 hours before operation. The dogs were anesthe-tized with pentobarbital (35 mg per kg iv). The following types of preparations were used: a) hepatecto-mized dogs, b) hepatectomized dogs with thoracic duct fistulas, c) eviscerated dogs, and d) an anesthetized normal control.

The uptake of the apoprotein and cholesteryl ester of high-density lipoproteins by the perfused rat liver

The uptake of the 125I-labeled apolipoprotein and 3H-labeled cholesteryl ester components of rat apolipoprotein E-deficient HDL by the perfused liver was studied. The uptake of the cholesteryl ester moiety was 4-fold higher than that of apolipoprotein. The concentration-dependent uptake of labeled protein was saturable and competed for by an excess of unlabeled HDL. The uptake of cholesteryl ester was not saturable over the concentration range studied. In the presence of a 50-fold excess of unlabeled HDL, the uptake of both radiolabeled components was decreased by over 75%, indicating that three-quarters of the hepatic uptake of HDL is by a receptor-mediated process. After 15 min of perfusion, 37% of the apolipoprotein radioactivity that was initially bound at 5 min was released into the perfusate as a more dense particle. After 5, 15, 30 and 60 min of perfusion the subcellular distribution of the apolipoprotein and cholesteryl ester components was analyzed by Percoll density gradient centrifugation. Over the 60 min period, there appeared to be transfer of radioactivity from the plasma membrane fraction to the lysosomal fraction. However, the internalization and degradation of cholesteryl ester was more rapid than that of the apolipoprotein. Our findings indicate that there is preferential uptake of HDL cholesteryl ester relative to protein by the liver and that the internalization of these components may occur independently.

The Clinical Significance of the Plasma High Density Lipoproteins

The consistent body of evidence supporting a relationship between high density lipoproteins and coronary heart disease is examined. However, an actual causal relationship has yet to be demonstrated. Methods of measuring HDL are presented and the difficulties with clinical measurement and evaluation are explored.

Protein-Lipid Relationships in Human Plasma. III. In Pregnancy and the Newborn

This study of pregnant women and their newborn infants was undertaken as a part of a more extensive investigation of factors that modify the interrelations of proteins and lipids in human plasma (1-3). The composition of maternal and fetal blood has been examined by many observers who, however, have usually focussed attention either on proteins or lipids. Electrophoretic studies of proteins have been made by Longsworth, Curtis, and Pembroke (4) who analyzed both serum and plasma; by Lagercrantz (5) and Moore, DuPan, and Buxton (6) who examined only serum; and by Macy and Mack (7) whose monograph was devoted to an inquiry concerning plasma proteins in human reproduction. There is consensus that maternal plasma has less than normal concentration of albumin and a greater than normal concentration of alpha and beta globulins while gammaglobulins are unchanged or only slightly decreased. These electrophoretic analyses of the cord plasma have shown lower than normal concentration of total protein which is chiefly attributable to a marked reduction in the concentration of alpha and beta globulins. Numerous investigators (8-10) have shown that in maternal blood the concentration of cholesterol and phospholipids is greater than normal while in blood from the umbilical cord at the time of birth it is notably reduced (11-13). In the present study, utilization of the microfractionation method No. 10 of Cohn and his coworkers (14) has made possible the separation of the lipoproteins of plasma into two fractions which together contain essentially all of the lipids. By