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.

Serum lipids and lipoproteins in advanced age. Intraindividual changes.

The Bronx Aging Study is a longitudinal investigation of nondemented, nonterminally ill, community-residing, old old volunteer subjects, designed to assess risk factors for the development of dementia and coronary and cerebrovascular diseases. During the first five annual evaluations, total cholesterol, high-density (HDL) and low-density lipoprotein (LDL), and triglyceride levels were measured. Mean cholesterol values (+/- standard error of the mean) for subjects at baseline were significantly higher for women than for men. Respectively, the values for total cholesterol were 6.1 +/- .1 mm/L (234 +/- 3 mg/dL) and 5.3 +/- .1 mm/L (207 +/- 3 mg/dL); for LDL cholesterol, 4.1 +/- .1 mm/L (158 +/- 2 mg/dL) and 3.7 +/- .1 mm/L (141 +/- 3 mg/dl); and for HDL cholesterol, 1.2 +/- .1 mm/L (47 +/- 1 mg/dL) and 1.0 +/- .1 mm/L (38 +/- 1 mg/dL). Mean triglyceride levels were 1.5 +/- .1 mm/L (135 +/- 5 mg/dL) for women and 1.6 +/- .1 mm/L (138 +/- 5 mg/dL) for men. Further, mean values remained stable over time. However, there was considerable intraindividual change observed in a substantial proportion of subjects between initial and final determinations. Changes of at least 10% from baseline were observed in 41%, 63%, 52%, and 78% of the cohort for cholesterol, HDL, LDL, and triglycerides, respectively. Thus, single measurements appear inadequate for establishing a diagnosis of hyperlipidemia in the elderly.

Plasma triglycerides and cholesterol in diabetic ketosis.

To better characterize the severity and course of hyperlipidemia in diabetic ketosis and ketoacidosis, we measured plasma triglyceride and cholesterol concentrations in 50 episodes in 46 adults hospitalized on a municipal hospital medical service. Moderate hypertriglyceridemia was common: 32 patients (64%) had triglyceride levels above the 95th percentile (adjusted for age and sex), and 18 patients (36%) had cholesterol levels above the 95th percentile. Severe hypertriglyceridemia (levels above 5.65 mmol/L) was found in 14 patients (28%). Plasma high-density lipoprotein (HDL) cholesterol and the HDL2 and HDL3 subclasses were measured in 22 episodes. The initial HDL cholesterol levels were usually subnormal in the patients who had not received insulin previously and normal in those who had. Treatment of the ketoacidosis was usually associated with a rapid decrease in plasma lipid levels. At late follow-up (between 1 1/2 and 14 1/2 months), only 2 of the 14 patients with initial plasma triglyceride levels above 5.65 mmol/L still had such high concentrations.

Hyperlipoproteinemia in spontaneously diabetic guinea pigs.

A colony of Hartley guinea pigs that exhibit hyperglycemia, glucosuria, and hypertriglyceridemia characteristic of human diabetes mellitus was developed. Initially, a group of guinea pigs that had normal serum glucose concentrations (less than or equal to 200 mg/dL of serum) at 3 to 4 weeks of age was obtained; however, in some of the animals progressively severe hyperglycemia (300 to 500 mg/dL of serum) and glucosuria (greater than 2 g of glucose/24 h) occurred as the animals matured. In addition, the animals exhibiting hyperglycemia and glucosuria had plasma insulin concentrations that were similar to those animals that were not hyperglycemic. The diabetic animals were found to be hypertriglyceridemic, with plasma triglyceride levels of 140 to 290 mg/dL at four months of age. Nondiabetic animals (plasma glucose concentration of less than or equal to 200 mg/dL and no glucosuria) had plasma triglyceride concentrations between 37 and 76 mg/dL. Lipoprotein analysis of plasma from nondiabetic and diabetic animals indicated that the diabetics had a fourfold increase in VLDL triglyceride and protein concentrations. The VLDL had an abnormal apolipoprotein composition and had reduced levels of apoprotein-E. The progeny from the mating of diabetic males and females also exhibited the diabetic trait, suggesting that the origin of the disease is genetic. This colony of guinea pigs is being further investigated as a suitable model for the study of the hyperlipoproteinemia of human noninsulin-dependent diabetes mellitus.

Determinants of the uptake of very low density lipoprotein remnants by the perfused rat liver.

The receptor-mediated uptake of very low density lipoprotein (VLDL) remnants by the rat liver was studied. Livers were perfused with native 125I-VLDL remnants, radiolabeled apo E-deficient remnants, and radiolabeled remnants that contained reductively methylated apo B and unmodified apo E. The specific uptake of the apo E-deficient remnants was 20% of that for the native remnants, whereas the specific uptake of the remnants containing unreactive apo B was 78% of the control value. This suggests that the apo E of VLDL remnants is the principal ligand for binding to the receptor, and in the absence of apo E, apo B may participate in binding. This conclusion is supported by the finding that dimyristoyl phosphatidylcholine (DMPC)- apo E complexes were effective in competing for the hepatic uptake of 125I-VLDL remnants. The intracellular distribution of radioactivity was analyzed by Percoll density gradient centrifugation. At five minutes after perfusion, radioactivity was associated with the plasma membrane and lysosomal fractions, and at 30 minutes most of the radioactivity was associated with the lysosomal fraction. Binding and internalization of VLDL remnants was also directly visualized by electron microscopy. Internalization proceeded by coated pit-coated vesicle formation with subsequent delivery to lysosomes. Our findings demonstrate that the apo E of VLDL remnants mediates binding to the hepatic receptor and that the internalization and degradation of VLDL remnants is by a similar pathway to that previously described for LDL.

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes.

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.

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.

High-density lipoprotein subclasses in diabetes.

This cross-sectional study evaluated high-density lipoprotein subclasses measured by a precipitation technique before and after treatment in men and women with types I and II diabetes. Total high-density lipoprotein cholesterol was lower in subjects of both sexes with untreated type I and type II diabetes, the change occurring primarily in subclass 2. Insulin therapy raised total and subclass 2 high-density lipoprotein levels in men and women with type I and type II diabetes, the predominant rise occurring in subclass 2. The improvement was unrelated to metabolic control. Normalization of total and subclass 2 high-density lipoprotein was not achieved in women with type II disease who had higher body weights and triglyceride levels. Treatment with oral hypoglycemic agents did not lower total high-density lipoprotein or subclass 2 levels. It is concluded that therapies affecting high-density lipoprotein produce the preponderant change in subclass 2, insulin therapy increases total and subclass 2 high-density lipoprotein but may not restore levels to normal in the presence of elevated body weight and lipid levels, there is no relation between control of diabetes and changes in total and subclass 2 high-density lipoprotein levels, and treatment with oral hypoglycemic agents does not adversely affect high-density lipoprotein levels.

The effect of glipizide on HDL and HDL subclasses.

The effects of glipizide on HDL subclass levels were prospectively evaluated in 7 women and 2 men with non-insulin dependent (Type 2) diabetes. Total HDL, HDL2 and HDL3 levels were unchanged during the treatment period. Baseline HDL levels were lower when compared to a control population which may have been due to the elevated body weight present in most subjects. Mean blood glucose and HbA1 levels were unchanged for the entire group although significant improvement was noted in 5 individuals. Triglyceride and cholesterol levels were not affected by treatment with glipizide. In conclusion, glipizide does not have an adverse affect on HDL lipoprotein levels when patients are followed prospectively.

Isolation and lipid-binding properties of rat apolipoprotein A-IV.

Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I.

A hepatocyte receptor for high-density lipoproteins specific for apolipoprotein A-I.

Apolipoprotein (apo) E-deficient rat high-density lipoproteins (HDL) bind to isolated rat hepatocytes at 4 degrees C by a process shown to be saturable and competed for by an excess of unlabeled HDL. Uptake (binding and internalization) at 37 degrees C was also saturable and competed for by an excess of unlabeled HDL. At 37 degrees C the HDL apoprotein was degraded as evidenced by the appearance of trichloroacetic acid-soluble radioactivity in the incubation media. The binding of a constant amount of 125I-apo-E-deficient HDL was measured in the presence of increasing concentrations of various lipoproteins. HDL and dimyristoyl phosphatidylcholine (DMPC) X apo-A-I complexes decreased binding by 80 and 65%, respectively. Human low-density lipoproteins, DMPC X apo-E complexes, and DMPC vesicles alone did not compete for apo-E-deficient HDL binding. However, DMPC X apo-E complexes did compete for the binding of the total HDL fraction that contained apo-E but to a lesser extent than did DMPC X apo-A-I. DMPC X 125I-apo-A-I complexes also bound to hepatocytes, and this binding was competed for by excess HDL (70%) and DMPC X apo-A-I complexes (65%), but there was no competition for binding by DMPC vesicles or DMPC X apo-E complexes. It thus appears that hepatocytes have a specific receptor for HDL and that apo-A-I is the ligand for this receptor.

Removal of lipid-rich lipoproteins by the liver.

Studies were performed to determine the mechanism of hepatic removal of a cholesterol-rich beta-migrating lipoprotein. This fraction, designated IDLc, was isolated from the serum of cholesterol-fed diabetic rats by ultracentrifugation at d 1.006-1.03 g/ml and contained apoproteins B, E, C, and A-I. When 125I-IDLc (125I-labeled IDLc) was injected into normal chow-fed rats, 40% of the radioactivity was cleared from the plasma within 5 minutes with slight additional removal during the next 25 minutes. The rapid removal phase was due to the clearance of apoB-containing lipoproteins. The slow removal phase was due to transfer of apoA-I and C-apoproteins to HDL which has a considerably slower rate of turnover. The in vivo clearance of total 125I-IDLc radioactivity was enhanced by pretreatment of normal rats with 17 alpha-ethinyl estradiol. This appeared to be associated with lack of transfer of apoA-I and C-apoproteins to HDL, and the removal of these apoproteins along with the apoB-containing lipoproteins. Treatment of rats with 17 alpha-ethinyl estradiol did not result in an increased rate of removal of 125I-IDLc when their livers were perfused and this suggests that the removal of IDLc is not mediated by the LDL (B, E) receptor whose activity is stimulated by estradiol administration.

Lipoproteins and coronary artery disease.

Although a great deal has been learned about HDL as a protective factor, our understanding of the role of HDL is far less comprehensive than that of LDL. Until such knowledge is available, efforts directed at altering HDL levels must be tentative and subject to revision, as indicated by experience. There is no evidence to warrant the use of drugs whose primary effect is to raise HDL.

Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.

Studies in recent years have suggested that measurement of high density lipoprotein (HDL) subclasses may provide significant information beyond that provided by measurement of total HDL. However, conventional methodology for separation of HDL subclasses involves various types of ultracentrifugation that are time-consuming, costly, and not suitable for many clinical or epidemiological studies. We have developed a simple precipitation method for the separation of HDL subclasses in human plasma. After precipitation of apoB-containing lipoproteins with heparin-Mn2+, HDL2 is precipitated by addition of dextran sulfate (mol wt 15,000). HDL2 cholesterol is calculated as the difference between total HDL cholesterol (heparin-Mn2+ supernatant) and HDL3 cholesterol (dextran sulfate supernatant). HDL2 determined by this method correlated well with results obtained by preparative ultracentrifugation (n = 295, r = 0.91) and analytical ultracentrifugation (n = 17, r = 0.92). In the original method final concentration of dextran sulfate was 0.09 g/dl; however further studies indicated that 0.13 g/dl is a more suitable concentration. The chemical compositions of HDL2 and HDL3 isolated by the precipitation method were very similar to those of HDL2 and HDL3 isolated by preparative ultracentrifugation. The concentration of HDL2 cholesterol was 40% higher in normal women than in normal men. In men with coronary heart disease, total HDL was decreased by 28%, HDL2 was decreased by 44%, while HDL3 was 19% lower. A similar pattern of change was found in women with coronary heart disease. In other conditions where total HDL either increased or decreased, the change in HDL2 was always proportionately greater than the change in total HDL. HDL3 showed relatively less change, and in some instances its concentration was unchanged. Thus HDL2 is the more variable component and may be a more meaningful index of altered HDL metabolism.