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New Zealand white rabbits exhibited as much as a threefold increase in plasma cholesterol but no change in hepatic cholesterol when fasted for 7–9 days. Agarose electrophoresis and ultracentrifugation of plasma samples showed that only low density lipoprotein increased during fasting. Fasting changed the composition of the low density lipoprotein by increasing the percentage of cholesterol and decreasing the percentage of triglyceride while protein and phospholipid remained the same. Rates of cholesterol secretion into plasma, measured by Triton WR 1339 injection, and rates of plasma cholesteryl ester synthesis, determined by [2-14C]mevalonate injection, were similar for fed and fasted rabbits. These findings suggest that fasting hypercholesterolemia in rabbits did not result from increased production of low density lipoproteins. Triton WR 1339 was shown to inhibit plasma cholesterol esterification in vitro.

Several methods for measuring cholesterol absorption in the rat have been compared. After administration of an oral dose of labeled cholesterol (14C or 3H) and an intravenous dose of colloidal labeled cholesterol (3H or 14C) the ratio of the two labels in plasma or whole blood 48 hr or more after dosing compared closely to the ratio of areas under the respective specific activity-time curves. The area ratio method is independent of a time lag between the appearance of oral and intravenous label in the bloodstream. Both measures of cholesterol absorption agree fairly well with a method based on measuring the unabsorbed dietary cholesterol in a pooled fecal sample. The plasma isotope ratio method gave more reproducible results than the fecal collection method when the measurement was repeated in the same animals 5 days after the first measurement. Cholesterol absorption was overestimated by the use of Tween 20-solubilized labeled cholesterol for the intravenous dose. The plasma disappearance curves of injected labeled colloidal cholesterol and cholesterol-labeled chylomicrons infused intravenously over a 3.5-hr period in the same animal coincided within experimental error from the first day until 75 days after injection. The plasma isotope ratio method for cholesterol absorption gave the same results in rats practicing coprophagy as in those in which this practice was prevented. The addition of sulfaguanidine to the diet lowered cholesterol absorption as measured by the plasma isotope ratio to the same degree as that measured by the fecal collection method.

Urinary and fecal endogenous steroid excretion of fed or fasted New Zealand white rabbits was determined by the isotopic steady state method after subcutaneous implantation of radioactive cholesterol. While plasma cholesterol was increasing during a 9-day fast, fecal steroid excretion decreased to 10% of the excretion rates in the fed state. Refeeding the fasted rabbits led to a decrease in plasma cholesterol and an increase in fecal endogenous steroid excretion. Urinary steroid excretion, which represented 18% of total endogenous steroid excretion for fed animals, decreased during fasting and increased during refeeding, but these changes were relatively small. The small intestine, cecum, and colon of fed or fasted rabbits had similar endogenous steroid was acidic steroid. During attempts to alter the circulating bile acid concentration by supplying deoxycholate (200 mg/day) to fed rabbits or cholestyramine (2 g/day) to fasted rabbits, plasma cholesterol concentration did not change to the same extent as during fasting or refeeding, respectively. The decreased cholesterol catabolism and the hypercholesterolemia that are seen in the fasting rabbit may result from decreased clearance of plasma cholesterol.

The daily administration of labeled cholesterol to humans or animals leads to an isotopic steady state. The specific activity of plasma cholesterol in the isotopic steady state gives information about the fraction of plasma cholesterol derived from endogenous and exogenous sources. A method, based on a two-pool model, is presented which allows the estimation of an optimal priming dose of labeled cholesterol whereby the time to reach the isotopic steady state is reduced to a minimum. A graphic procedure is presented which allows the estimation of an optimal priming dose for two-compartment systems with widely differing characteristics.

An improved separation of phospholipids on activated silicic-acid columns was achieved by stepwise elution with increasing concentrations of methanol in chloroform. Chemical analyses and paper strip chromatography demonstrated that the noncholine-containing fraction was free of choline-containing phospholipids, and that the lecithin and lysolecithin were of high purity. Rat plasma was shown to contain on the average 17.5% of its lipid phosphorus in the form of lysolecithin. The following is evidence that plasma lysolecithin is not an in vitro breakdown product: (a) rapid glass-paper chromatography of lipid extracts showed the presence of a distinct spot with the Rf of lysolecithin, (b) after P32 injection the specific activity of lysolecithin was less than that of lecithin, (c) silicic-acid chromatography of C14-labeled plasma lipids failed to show a significant liberation of fatty acids, and (d) rechromatography of P32-labeled lecithin did not give rise to a lysolecithin peak.

Dog lymph chylomicrons incubated with serum showed a progressive gain in free cholesterol whereas no change took place in the cholesterol ester and triglyceride fractions. Chylomicron phospholipids were observed to suffer a net loss while at the same time exchanging with serum phospholipids. Before incubation, lymph chylomicron phospholipids contained a higher proportion of noncholine phospholipids than serum; during incubation, this fraction was largely transferred to serum. The noncholine fraction showed comparatively little exchange. Lecithin was by far the largest component of both chylomicron and serum phospholipids. In absolute amounts, the largest transfer and exchange could be accounted for by lecithin. The percentages of sphingomyelin and lysolecithin in chylomicron phospholipids were lower than in those of serum. During incubation, the sphingomyelin content of chylomicrons changed little, but active exchange of sphingomyelin took place. The amount of lysolecithin increased both in chylomicrons and in serum. Almost all the phospholipid lost from the chylomicrons appeared in the high-density lipoprotein fraction. Evidence is presented that the extra phospholipid is taken up by preexisting high-density lipoprotein. Exchange of phospholipids between chylomicrons and serum lipoprotein fractions took place about in proportion to the relative phospholipid contents of these fractions. The bearing of these results on the mechanisms whereby chylomicrons are removed from the circulation is discussed.

Unesterified radioactive cholesterol, both bound to serum lipoproteins and dispersed in ethanol–saline, was injected into bile fistula and intact rats. Due to phagocytosis, mainly by the liver macrophages, intravenously injected cholesterol in ethanol–saline disappears from the bloodstream significantly faster than lipoprotein-bound cholesterol.Soon after the initial phagocytosis, the particulate isotopic cholesterol started to reappear in blood, reaching a maximal radioactivity in blood 10–24 hr after injection. Although the radioactive cholesterol reappears in serum in both esterified and unesterified form, it is likely that cholesterol is released from the phagocytic cells as unesterified cholesterol which is then esterified intravascularly or at other sites. In the bile fistula rats, somewhat more of the lipoprotein cholesterol than of the particulate cholesterol appeared in bile early after injection. However, cholesterol turnover calculated from a twopool model was the same for rats injected with lipoprotein-bound or particulate cholesterol.

Rabbits fed cholesterol for 1 to 5 months showed gradually increasing amounts of aortic phospholipid preceded by increases in plasma phospholipid concentration. Cholesterol feeding appeared to increase the turnover of plasma as well as aortic phospholipid. Curtailment of plasma phospholipid synthesis to one-tenth of normal by evisceration did not diminish the incorporation of P32 into aortic lipids. In these animals the aortic phospholipid specific activity was 15 to 90 times as great as that of plasma, so that practically all the labeled aortic phospholipid must have been synthesized in situ. Lowering the plasma lipid levels by removal of cholesterol from the diet did not diminish the cholesterol content of the aortic lesion or alter its phospholipogenesis.

New Zealand white rabbits showed large decreases in plasma cholesterol and phospholipid concentrations during the second half of pregnancy. All lipoproteins (very low density, low density, and high density) participated in the decrease. Very large decreases in plasma cholesterol concentrations were observed even when the animals were maintained on high cholesterol diets. Increases in plasma cholesterol concentrations, after the intravenous administration of Triton WR 1339, were at least as great in pregnant as in nonpregnant animals. It is concluded that the decrease in plasma cholesterol concentrations is not the result of impaired plasma lipoprotein production.

Dogs fed a diet containing butter, cholesterol, and thiouracil develop severe hypercholesterolemia. Both α2- and β-lipoprotein cholesterol and phospholipid increase whereas these lipids in the α1 fraction appear to decrease. Comparison of the dextran sulfate-precipitable lipoproteins with lipoprotein fractions obtained by ultracentrifugation at densities 1.019, 1.063, and 1.21 and with paper electrophoretograms reveals that the addition of low molecular weight dextran sulfate and calcium chloride to diluted hypercholesterolemic dog serum quantitatively precipitates low-density lipoproteins. Paper electrophoresis revealed that the dextran sulfate-precipitable fraction contains both α2- and β-lipoproteins.

Serum high- and low-density lipoproteins were spread between heptane and phosphate buffer. The cholesterol from both protein films became completely soluble in the heptane phase. Other neutral lipids were also found in the heptane, but phospholipids could not be detected. Shaking of whole serum or isolated lipoproteins with heptane resulted in only slight extraction of cholesterol by the fat solvent. The addition of albumin to the lipoproteins reduced the amount of cholesterol extractable by heptane. It is postulated that when aqueous solutions of lipoproteins are shaken with nonpolar fat solvents the degree of cholesterol extraction depends on the disruption of the lipoprotein structure through contact with various interfaces, and that other proteins may protect the lipoprotein by competing for position at the interface.

Isolated livers perfused with washed corn oil chylomicrons labeled in vivo with palmitic acid-1-14C removed a large proportion of the chylomicrons. Slices from these livers oxidized chylomicron fatty acid esters to both carbon dioxide and acetoacetate. The liver slices also generated free fatty acids from chylomicron lipids and converted chylomicron triglycerides to phospholipids. Similar activities were observed in rat liver slices prepared shortly after the intravenous administration of chylomicrons to intact rats.The observed chylomicron uptake and lipid conversions were similar in livers from both fed and fasted rats. Fasting increased the oxidation of chylomicron fatty acid esters by livers labeled in vivo and by perfusion.In livers removed from intact rats given labeled chylomicrons, the triglyceride-14C to phospholipid-14C ratio was high, a finding unexpected if the liver had acquired this 14C by removal of circulating fatty acids formed by extrahepatic lipolysis.These results demonstrate the ability of the liver to remove and utilize chylomicrons directly and suggest that direct removal accounts for a significant portion of the chylomicron fatty acids utilized by the liver of intact rats.

Ponies fasted for up to 8 days showed, both by agarose electrophoresis and preparative ultracentrifugation, the appearance of a pre-β-migrating, very low density lipoprotein fraction in plasma. This lipoprotein differs from the very low density lipoprotein found in humans and rats in that it contains a relatively smaller amount of total cholesterol, 85% of which is present in the unesterified form. By the 8th day of fasting, plasma triglyceride concentrations had increased from a prefasting level of 20 mg/dl to as high as 1000 mg/dl. The increase in plasma lipid concentrations as a result of fasting was highly variable. Accumulation of plasma cholesterol and triglyceride after injection of Triton WR 1339 was not related to the degree of fasting hyperlipidemia. This suggests that the hyperlipoproteinemia of fasting may result from an impaired utilization of very low density lipoproteins.

Chyle from the thoracic duct of dogs fed cream or corn oil was fractionated into chylomicrons of different particle size: 200 mμ. The composition of chylomicrons was studied before and after washing with saline solutions. In general the percentages of protein, phospholipid, cholesterol, and cholesterol ester were greater in the small chylomicrons than in the larger ones.Washing the chylomicrons did not appear to affect their composition. Chylomicrons collected at different times during fat feeding showed significant differences in phospholipid and cholesterol concentrations. Storage of lymph at 4° was accompanied by alterations in phospholipid and cholesterol concentrations of the chylomicrons. The composition of chylomicrons is consistent with the hypothesis that at some stage during their formation the nontriglyceride components are present on the surface of the oil droplet.

Rat and guinea pig liver microsomes labeled with phospholipid 32P were incubated with rat, guinea pig, and rabbit plasma in a KC1–Tris–EDTA buffer. A net transfer of microsomal phosphatidylcholine and phosphatidylethanolamine to plasma was observed. In addition, an exchange of phospholipids between microsomes and plasma took place. During 20-min incubations at 37°C, the exchange of phosphatidylcholine was the most extensive. Microsomal sphingomyelin exchanged with plasma sphingomyelin only very slowly. A soluble protein factor in liver, which had previously been observed to stimulate the exchange of liver mitochondrial and microsomal phospholipids, also increased the exchange of phosphatidylcholine between liver microsomes and plasma. The pronounced differences in the relative percentages of phosphatidylethanolamine of guinea pig, rabbit, and rat plasmas did not appear to be related to differences in the relative exchange of this phospholipid compared to that of other phospholipids in these plasmas.

Electron microscope studies were performed on thoracic duct lymph and on washed chylomicrons from dogs fed corn oil. High-resolution electron micrographs showed the presence of a surface coat that differed from the core material and did not resemble a plasma membrane. This was true for both chylomicrons in whole lymph and those that had been subjected to repeated washing. Apparently, the chylomicrons, while passing from the intracellular to the extracellular space, do not acquire their surface coat from pinched off cellular membrane.

The separation of dog serum lipoproteins by ultracentrifugation at a density of 1.063 is hindered by the failure of the high density fraction to accumulate in the bottom portion of the centrifuge tube. This phenomenon interferes with the quantitative recovery of pure low density lipoproteins. By paper electrophoresis, a lipid-containing protein with the mobility of a β-globulin was detected in the lowest layer of the centrifuge tube. Comparison of the preparative ultracentrifuge technique with that of dextran sulfate precipitation of β-lipoproteins revealed the suit ability of the latter procedure for the quantitative separation of β-lipoproteins. The dextran sulfate β-lipoprotein precipitate was shown to be free from α-lipoprotein by paper electrophoresis.

Chylomicrons from the thoracic duct lymph of dogs fed corn oil were isolated by centrifugation and disrupted by either freezing and thawing or rotary evaporation and rehydration. A pellet, representing the surface coat, was isolated by centrifugation. Pellets isolated by freezing and thawing contained a higher percentage of saturated triglycerides than pellets isolated by rotary evaporation; the presence of saturated triglyceride in the pellet was probably an artifact of the preparation of the surface coat material at low temperature.Exchange of free cholesterol between surface and core lipid of chylomicrons was complete within 1 hr. The percentage of cholesterol in pellets of surface material isolated by freezing and thawing was about twice that found for pellets after rotary evaporation at 25-40°C. Cholesteryl ester was not present in the surface lipid and that present in the core lipid did not exchange with serum lipoprotein cholesteryl ester.For phosphatidyl choline, the percentage of linoleic acid in lymph chylomicrons was markedly higher than that in clear lymph or plasma, while the percentage of arachidonic acid was lower. Sphingomyelin of lymph chylomicrons was characterized by very high levels of 16 : 0 and relatively small percentages of very long-chain fatty acids as compared with clear lymph or plasma.The data are consistent with the view that in lymph chylomicrons: (a) cholesteryl esters are dissolved in a core of triglycerides which contain fatty acids derived primarily from dietary fatty acids, (b) free cholesterol is partitioned between core and surface and is freely exchangeable between the two, (c) the phospholipid fractions are present on the surface and are intracellular in origin.