Charles E. Day

Distribution of serum lipoproteins of selected vertebrates.

Abstract 1. 1. Agarose gel electrophoresis was performed on the sera of thirty-six mammals, reptiles, amphibians, fish and birds, and the quantitative distribution of the lipoprotein classes in each animal was determined. 2. 2. Pig, opossum and garter snake sera seem to contain large amounts of low density lipoprotein, the lipoprotein class which is of primary interest in atherosclerosis research. These animals also have serum cholesterol values similar to humans. 3. 3. Agarose gel electrophoresis proved an excellent method for determining lipoprotein distributions, but the electrophoretic mobilities of the lipoproteins appear to be unsuitable for lipoprotein classification.

High Volume Screening Procedures for Hypobetalipoproteinemic Activity in Rats

We describe high volume screening tests for hypobetalipoproteinemic agents in which compounds are administered orally to cholesterol-cholic acid fed (hypercholesterolemic( or normally fed weanling rats for 4 days. In these tests total serum cholesterol levels and heparin precipitating lipoproteins (HPL) are determined by automated analyses interfaced with a computer which eliminates all manual data reduction and provides necessary reports. The hypercholesterolemic rat test detects compounds which specifically reduce HPL (beta and pre beta lipoproteins) causing a decrease in the HPL: cholesterol ratio. Such activity is called hypobetalipoproteinemia. This activity is exhibited by bicyclo (2.2.2)-octyloxyaniline (U-26328) but not by any of the familiar hypocholesterolemic agents including clofibrats, lifibrats, nicotinic acid, probucol, triparanol, lentysine, D-throxine or the estrogens estrone and diethylstilbestrol.

Produced by selective breeding of Japanese quail animal model for experimental atherosclerosis.

The Japanese quail (Coturnix coturnix japonica) was bred selectively to produce a strain highly susceptible to experimental atherosclerosis. A population was produced where aortic atherosclerosis is contracted by 99% of the fourth generation males and 83% of the femlaes. Forty-three percent of the males exhibited severe atherosclerosis making this line of Japanese quail a suitable model for discovering and testing anti-atherosclerosis compounds. This feature is augmented by other features such as size, disposition, and abundance which qualify them as suitable experimental subjects. A second line of Japanese quail was bred to be resistant to dietary-induced atherosclerosis. This strain may be a useful research tool for characterizing the etiology of atherosclerosis.

Effects of tetracycline on ultrastructure and lipoprotein secretion in the rat hepatocyte

Abstract Fat droplets, 0.1–0.5 μm, accumulated in the space of Disse of hepatocytes in rats treated ip with 300 mg/kg of tetracycline hydrochloride. This change occurred in both pregnant and unbred females and in males but was not observed in similar rats treated with 100 mg/kg which showed typical steatosis induced by tetracycline. Ultrastructural changes in mitochondria, rough and smooth endoplasmic reticulum (SER) were similar to those previously reported. Uptake of iv administered palmitic [9-10-3H]acid in rats treated with 300 mg/kg was not appreciably impaired. One hour after an injection of 300 mg/kg, the antibiotic concentration was 10 times greater in the liver than in the serum. From 1 to 6 hr the concentrations increased from roughly 300 to 800 μg/g of liver. In rats treated with 100 mg/kg, no concentrations above 200 μg/g were observed during a 48-hr postinjection period. Under in vitro conditions, tetracycline complexed with lipoprotein in a precipitate when a concentration greater than 200 μg/ml was obtained in rat blood serum. In the hepatocyte of rats treated with 300 mg/kg it was presumed that as the antibiotic concentration rose above 200 μg/g nascent very low density lipoprotein complexed rapidly with it. The complexed lipoprotein was channeled in dilated SER in the manner of liposomes to the sinusoidal surface and discharged from vesicles into the space of Disse. The plasma membrane of the hepatocyte showed loss of microvilli and surface distortion. The space of Disse became distended with insoluble fat droplets from 6 hr to 3 days after injection.

New Animal Model for Atherosclerosis Research

Japanese quail were investigated for their utility as a model for the discovery and evaluation of anti-atherosclerosis compounds. Although they possessed suitable characteristics for a screening animal, their development of atherosclerosis was too variable to make them a practical model. A search was conducted to find a means to make quail uniformly atherosclerotic. To this end a line of quail susceptible to experimental atherosclerosis (SEA) were selectively bred. Thus, the SEA Japanese quail is a new animal model for atherosclerosis research.

Comparative Electrophoretic Profiles of Serum Lipoproteins

Reduction of hyperlipoproteinemias is accepted almost universally as one way to retard atherogenesis. An enormous research and development effort is expended each year in the search for hypolipidemic drugs. Rats or mice are generally used in the primary screening and subsequent follow-up testing for hypolipoproteinemic agents. But the lipoproteins of both rats and mice are quite different quantitatively and qualitatively from human lipoproteins. Clearly better animal models simulating the human lipoprotein spectrum are needed for atherosclerosis research, especially in the pharmaceutical industry. The primary requirement for such an animal model is an elevated low density lipoprotein (LDL) level and a high LDL/HDL ratio. Secondary considerations include size, cost, and ease of handling and maintenance of the animal. Size and cost are major considerations for drug screening, since compounds are usually available in only very small quantities and large numbers of animals are required. The purpose of this investigation was to survey the electrophoretic profiles of serum lipoproteins of several animals in an effort to discover a better animal model for human serum lipoproteins suitable for drug evaluations.

Evaluation of timefurone, a new anti-atherosclerotic drug, for its effects on lipoprotein cholesterol in male SEA Japanese quail and rats.

Timefurone was evaluated in several animal models for cholesterol-lowering and anti-atherosclerotic activity. In normal male rats, a dose-response study with timefurone (3, 10, 30, 50 and 100 mg/kg/day) was conducted for 7 days. Significant activity was observed only at 50 and 100 mg/kg/day, where very low and low density lipoprotein cholesterol [(VLDL + LDL)-C] and total-C levels were reduced (mean 27 and 20%). High density lipoprotein cholesterol (HDL-C) was lowered 24% by the high timefurone dose. Timefurone (10, 20, 50 and 100 mg/kg/day in the diet) was then examined in normocholesterolemic SEA japanese quail. beta-lipoprotein cholesterol (VLDL + LDL)-C was reduced at all doses (mean 58%), while alpha-lipoprotein cholesterol (HDL-C) was elevated by all doses of timefurone (mean 45%). Male weanling rats made moderately hypercholesterolemic represented a 3rd phase of timefurone (2.5, 5, 10, 20, 50, 100 mg/kg/day) testing. After 4 days of drug treatment, marked hypocholesterolemic activity was observed for (VLDL + LDL)-C (mean decrease 49%) and total-C (mean 33%). HDL-C levels were increased with 10 and 100 mg/kg/day doses. Timefurone (25 and 100 mg/kg/day in the diet) also caused a significant reduction in atherosclerotic development in hypercholesterolemic SEA japanese quail. Atherosclerotic involvement (determined by visual assessment of plaque), arterial weight, and arterial cholesterol (total and mg/g artery) were clearly lowered by both doses of timefurone. There was no evidence of significant drug toxicity in any of these experiments. On the basis of these data, timefurone has excellent therapeutic potential and additional study of the drugs hypocholesterolemic and anti-atherosclerotic properties appears warranted.

Response to the hypobetalipoproteinemic agent adamantyloxyphenyl piperidine in hyperlipemic rats.

The hypobetalipoproteinemic activity of U-41, 792 (1-[p-(1-adamantyloxy)-phenyl]-piperidine) is a marked and selective reduction of heparin precipitating lipoproteins (low density plus very low density lipoproteins) in cholesterol-cholic acid induced hypercholesterolemic rats. This activity consists of both a reduction in heparin precipitating lipoproteins (HPL) and an increase in high density lipoproteins that are not precipitated by heparin. The increase in high density lipoproteins is routinely noted by decreases in HPL/cholesterol ratios. The pattern of response following single 100 mg/kg doses of U-41, 792 was determined. After an I.V. dose was administered in a cotton-seed oil emulsion, serum cholesterol levels were reduced, beginning at 8 hr after administration and persisting for 96 hr. Similar results, though delayed somewhat, were obtained after a single oral dose. Activity was accompanied by increases in weight and cholesterol content of livers. After multiple, daily, oral doses, liver weights, total lipids, and cholesterol contents were reduced. Hypobetalipoproteinemic activity was enhanced by prolonged treatments as demonstrated by analyses of serum obtained weekly throughout 7 wk.

CONTROL OF THE INTERACTION OF CHOLESTEROL ESTER-RICH LIPOPROTEINS WITH ARTERIAL RECEPTORS

Incorporation of 125I-labeled cholesterol ester rich lipoproteins from cholesterol fed rabbits into normal rabbit aorta in vitro was inhibited by heparin, lecithin, and collagenase and by succinylation of the lipoprotein. Aortic uptake of lipoprotein was increased by neuraminidase, proteases, lipase, and beta-glucuronidase. These results suggest that it may be possible to control atherogenesis by controlling the interaction of atherogenic lipoproteins with their arterial receptor.

Composition of very low density lipoproteins from cholesterol fed animals

1. 1. Very low density lipoproteins (VLDL, d < 1·006 g/ml) were isolated from the sera of cholesterol fed rabbits, pigeons and Japanese quail and from cholesterol-cholic acid fed rats. 2. 2. Collectively, the composition of these lipoproteins was 10 per cent protein, 61 per cent cholesterol, 8 per cent triglyceride and 21 per cent phospholipid. More than half (50–75 per cent) of the total serum cholesterol was in the VLDL. 3. 3. After comparing their composition with normal and abnormal human and animal lipoproteins, we conclude that VLDL from cholesterol fed animals have a distinct and unique composition based largely on their very high content of cholesterol esters (CE). Therefore, this lipoprotein can be called appropriately CE-VLDL.